Novel antimicrobial agents

ABSTRACT

A novel class of antimicrobial polymeric agents, which include a plurality of positively charged amino acid residues and at least one omega-amino-fatty acid residue linking therebetween, designed to exert antimicrobial activity while being stable, non-toxic and avoiding development of resistance thereto and a process of preparing same are disclosed. Further disclosed are pharmaceutical compositions containing same and a method of treating medical conditions associated with pathological microorganisms, a medical device, an imaging probe and a food preservative utilizing same. Further disclosed are conjugates of an amino acid residue and a hydrophobic moiety residue and a process of preparing same.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/819,486 filed Aug. 6, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/031,295 filed Feb. 21, 2011, which iscontinuation of U.S. patent application Ser. No. 11/500,461 filed Aug.8, 2006, now U.S. Pat. No. 7,915,223, which is Continuation-in-Part(CIP) of U.S. patent application Ser. No. 11/234,183 filed Sep. 26,2005, now U.S. Pat. No. 7,504,381, which claims the benefit of priorityof U.S. Provisional Patent Application No. 60/162,778 filed Sep. 27,2004. The contents of the above applications are incorporated herein byreference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel antimicrobial agents and, moreparticularly, to a novel class of polymers which are designed to exertantimicrobial activity while being stable, non-toxic and avoidingdevelopment of resistance thereto. The present invention further relatesto pharmaceutical compositions, medical devices and food preservativescontaining such polymers and to methods of treating medical conditionsassociated with pathogenic microorganisms utilizing same.

Antibiotics, which are also referred to herein and in the art asantibacterial or antimicrobial agents, are natural substances ofrelatively small size in molecular terms, which are typically releasedby bacteria or fungi. These natural substances, as well as derivativesand/or modifications thereof, are used for many years as medications fortreating infections caused by bacteria.

As early as 1928, Sir Alexander Fleming observed that colonies of thebacterium Staphylococcus aureus could be destroyed by the moldPenicillium notatum. His observations lead Fleming to postulate theexistence and principle of action of antibiotic substances. It wasestablished that the fungus releases the substance as a mean ofinhibiting other organisms in a chemical warfare of microscopic scale.This principle was later utilized for developing medicaments that killcertain types of disease-causing bacteria inside the body. In 1940'sHoward Florey and Ernst Chain isolated the active ingredient penicillinand developed a powdery form of the medicine.

These advancements had transformed medical care and dramatically reducedillness and death from infectious diseases. However, over the decades,almost all the prominent infection-causing bacterial strains havedeveloped resistance to antibiotics.

Antibiotic resistance can result in severe adverse outcomes, such asincreased mortality, morbidity and medical care costs for patientssuffering from common infections, once easily treatable with antibiotics(Am. J. Infect. Control 24 (1996), 380-388; Am. J. Infect. Control 27(1999), 520-532; Acar, J. F. (1997), Clin. Infect. Dis. 24, Suppl 1,S17-S18; Cohen, M. L. (1992), Science 257, 1050-1055; Cosgrove, S. E.and Carmeli, Y. (2003), Clin. Infect. Dis. 36, 1433-1437; Holmberg, S.D. et al. (1987), Rev. Infect. Dis. 9, 1065-1078) and therefore becameone of the most recognized clinical problems of today's governmental,medicinal and pharmaceutical research (U.S. Congress, Office ofTechnology Assessment, Impacts of Antibiotic-Resistant Bacteria,OTA-H-629, Washington, DC, U.S. Government Printing Office (1995); Houseof Lords, Science and Technology 7th Report: Resistance to Antibioticsand Other Antimicrobial Agents, HL Paper 81-II, session (1997-98); andInteragency Task Force on Antimicrobial Resistance, A Public HealthAction Plan to Combat Antimicrobial Resistance. Part 1: Domesticissues).

Due to the limitations associated with the use of classical antibiotics,extensive studies have been focused on finding novel, efficient andnon-resistance inducing antimicrobial/antibacterial agents.

Within these studies, a novel class of short, naturally occurringpeptides, which exert outstanding antimicrobial/antibacterial activity,was uncovered.

These peptides, which are known as antimicrobial peptides (AMPs), arederived from animal sources and constitute a large and diverse family ofpeptides, which may serve as effective antimicrobial agents againstantibiotic-resistant microorganisms (for recent reviews see, forexample, Levy, O. (2000) Blood 96, 2564-2572; Mor, A. (2000) DrugDevelopment Research 50, 440-447; Zasloff, M. (2002) New England Journalof Medicine 347, 1199-1200; Zasloff, M. (2002) Nature 415, 389-395;Zasloff, M. (2002) Lancet 360, 1116-1117). In the past 20 years, over700 AMPs derived from various sources, from unicellular organisms tomammalians and including humans, have been identified (for recentreviews see, for example, Andreu, D. and Rivas, L. (1998) Biopolymers47, 415-433; Boman, H. G. (2003) J. Intern. Med. 254, 197-215; Devine,D. A. and Hancock, R. E. (2002) Curr. Pharm. Des. 8, 703-714; Hancock,R. E. and Lehrer, R. (1998) Trends Biotechnol. 16, 82-88; Hancock, R. E.(2001) Lancet Infect. Dis. 1, 156-164; Hancock, R. E. and Rozek, A.(2002) FEMS Microbiol. Lett. 206, 143-149; Hoffmann, J. A. andReichhart, J. M. (2002) Nat. Immunol. 3, 121-126; Lehrer, R. I. andGanz, T. (1999) Curr. Opin. Immunol. 11, 23-27; Nicolas, P. and Mor, A.(1995) Annu. Rev. Microbiol. 49, 277-304; Nizet, V. and Gallo, R. L.(2002) Trends Microbiol. 10, 358-359; Shai, Y. (2002) Curr. Pharm. Des.8, 715-725; Simmaco, M. et al. (1998) Biopolymers 47, 435-450; Tossi, A.et al. (2000) Biopolymers 55, 4-30; Tossi, A. and Sandri, L. (2002)Curr. Pharm. Des. 8, 743-761; Vizioli, J. and Salzet, M. (2002) TrendsPharmacol. Sci. 23, 494-496; Brogden, K. et al. (2003) Int. J.Antimicrob. Agents 22, 465-478 and Papagianni, M. (2003) Biotechnol.Adv. 21, 465-499).

AMPs are now recognized to have an important role in the innate hostdefense. They display a large heterogeneity in primary and secondarystructures but share common features such as amphiphatic character andnet positive charge. These features appear to form the basis for theircytolytic function. Ample data indicate that AMPs cause cells death bydestabilizing the ordered structure of the cell membranes, although thedetailed mechanism has not been fully understood yet (for recent reviewssee, for example, Epand, R. M. et al. (1995), Biopolymers 37, 319-338;Epand, R. M. and Vogel, H. J. (1999), Biochim. Biophys. Acta 1462,11-28; Gallo, R. L. and Huttner, K. M. (1998), J. Invest Dermatol. 111,739-743; Gennaro, R. et al. (2002), Curr. Pharm. Des. 8, 763-778;Hansen, J. N. (1994), Crit Rev. Food Sci. Nutr. 34, 69-93; Huang, H. W.(1999), Novartis. Found. Symp. 225, 188-200; Hwang, P. M. and Vogel, H.J. (1998), Biochem. Cell Biol. 76, 235-246; Lehrer, R. I. et al. (1993),Annu. Rev. Immunol. 11, 105-128; Matsuzaki, K. (1999), Biochim. Biophys.Acta 1462, 1-10; Muller, F. M. et al. (1999), Mycoses 42 Suppl 2, 77-82;Nissen-Meyer, J. and Nes, I. F. (1997), Arch. Microbiol. 167, 67-77;Peschel, A. (2002), Trends Microbiol. 10, 179-186; Sahl, H. G. andBierbaum, G. (1998), Annu. Rev. Microbiol. 52, 41-79; Shai, Y. (1995),Trends Biochem. Sci. 20, 460-464; and Yeaman, M. R. and Yount, N. Y.(2003), Pharmacol. Rev. 55, 27-55). It is assumed that disturbance inmembrane structure leads to leakage of small solutes (for example K⁺,amino acids and ATP) rapidly depleting the proton motive force, starvingcells of energy and causing cessation of certain biosynthetic processes(Sahl, H. G. and Bierbaum, G. (1998), Annu. Rev. Microbiol. 52, 41-79).This mechanism is consistent with the hypothesis that antimicrobialactivity is not mediated by interaction with a chiral center and maythus significantly prevent antibiotic-resistance by circumventing manyof the mechanisms known to induce resistance.

In addition to their direct well-documented cytolytic(membrane-disrupting) activity, AMPs also display a variety ofinteresting biological activities in various antimicrobial fields. SomeAMPs were shown to activate microbicidal activity in cells of the innateimmunity including leukocytes and monocyte/macrophages (Ammar, B. et al.(1998), Biochem. Biophys. Res. Commun. 247, 870-875; Salzet, M. (2002)Trends Immunol. 23, 283-284; Scott, M. G. et al. (2000), J. Immunol.165, 3358-3365; and Scott, M. G. et al. (2002), J. Immunol. 169,3883-3891). Many cationic peptides are endowed with lipopolysaccharidebinding activity, thus suppress the production of inflammatory cytokinesand protect from the cascade of events that leads to endotoxic shock(Chapple, D. S. et al. (1998), Infect. Immun. 66, 2434-2440; Elsbach, P.and Weiss, J. (1998), Curr. Opin. Immunol. 10, 45-49; Lee, W. J. et al.(1998), Infect. Immun. 66, 1421-1426; Giacometti, A. et al. (2003), J.Chemother. 15, 129-133; Gough, M. et al. (1996), Infect. Immun. 64,4922-4927; and Hancock, R. E. and Chapple, D. S. (1999), Antimicrob.Agents Chemother. 43, 1317-1323). Antimicrobial genes introduced intothe genome of plants granted the plant the resistance to pathogens byexpressing the peptide (Alan, A. R. et al. (2004), Plant Cell Rep. 22,388-396; DeGray, G. et al. (2001), Plant Physiol 127, 852-862; Fritig,B., Heitz, T. and Legrand, M. (1998), Curr. Opin. Immunol. 10, 16-22;Osusky, M. et al. (2000), Nat. Biotechnol. 18, 1162-1166; Osusky, M. etal. (2004), Transgenic Res. 13, 181-190; and Powell, W. A. et al.(2000), Lett. Appl. Microbiol. 31, 163-168).

On top of the ribosomally synthesized antimicrobial peptides that havebeen identified and studied during the last 20 years, thousands ofde-novo designed AMPs, were developed (Tossi, A. et al. (2000),Biopolymers 55, 4-30). These de-novo designed peptides are comprised ofartificially designed sequences and were produced by genetic engineeringor by chemical peptide syntheses. The finding that various antimicrobialpeptides, having variable lengths and sequences, are all active atsimilar concentrations, has suggested a general mechanism for theanti-bacterial activity thereof rather than a specific mechanism thatrequires preferred active structures (Shai, Y. (2002), Biopolymers 66,236-248). Naturally occurring peptides, and de-novo peptides havingartificially designed sequences, either synthesized by humans orgenetically engineered to be expressed in organisms, exhibit variouslevels of antibacterial and antifungal activity as well as lyticactivity toward mammalian cells. As a result, AMPs are attractivetargets for bio-mimicry and peptidomimetic development, as reproductionof critical peptide biophysical characteristics in an unnatural,sequence-specific oligomer should presumably be sufficient to endowantibacterial efficacy, while circumventing the limitations associatedwith peptide pharmaceuticals (Latham, P. W. (1999), Nat. Biotechnol. 17,755-757).

One of the challenges in designing new antimicrobial peptides relies ondeveloping peptidomimetics that would have high specificity towardbacterial or fungal cells, and consequently, would allow betterunderstanding of the mechanism underlying the peptide lytic specificity,i.e., discrimination between cell membranes. Structure-activityrelationships (SAR) studies on AMPs typically involve the systematicmodification of naturally occurring molecules or the de-novo design ofmodel peptidomimetics predicted to form amphiphatic alpha-helices orbeta-sheets, and the determination of structure and activity via variousapproaches (Tossi, A. et al. (2000), Biopolymers 55, 4-30), as follows:

Minimalist methods for designing de-novo peptides are based on therequirement for an amphiphatic, alpha-helical or beta-sheet structure.The types of residues used are generally limited to the basic,positively charged amino acids lysine or arginine, and one to three ofthe hydrophobic residues alanine, leucine, isoleucine, glycine, valine,phenylalanine, or tryptophan (Blazyk, J. et al. (2001), J. Biol. Chem.276, 27899-27906; Epand, R. F. et al. (2003), Biopolymers 71, 2-16;Hong, J. et al. (1999), Biochemistry 38, 16963-16973; Jing, W. et al.(2003), J. Pept. Res. 61, 219-229; Ono, S. et al. (1990), Biochim.Biophys. Acta 1022, 237-244; and Stark, M. et al. (2002), Antimicrob.Agents Chemother. 46, 3585-3590). While these approaches may lead to thedesign of potent antimicrobial agents, subtleties to the sequence ofAMPs that may have been selected for by evolution are not considered andtheir absence may lead to a loss of specificity.

Sequence template methods for designing and synthesizing amphiphaticAMPs typically consists of extracting sequence patterns after comparisonof a large series of natural counterparts. The advantage of this method,as compared with conventional sequence modification methods, is that itreduces the number of peptides that need to be synthesized in order toobtain useful results, while maintaining at least some of the sequencebased information. As discussed hereinabove, the latter is lost inminimalist approaches (Tiozzo, E. et al. (1998), Biochem. Biophys. Res.Commun. 249, 202-206).

Sequence modification method includes all of the known and acceptablemethods for modifying natural peptides, e.g., by removing, adding, orreplacing one or more residues, truncating peptides at the N- orC-termini, or assembling chimeric peptides from segments of differentnatural peptides. These modifications have been extensively applied inthe study of dermaseptins, cecropins, magainins, and melittins inparticular (Scott, M. G. et al. (2000), J. Immunol. 165, 3358-3365;Balaban, N. et al. (2004), Antimicrob. Agents Chemother. 48, 2544-2550;Coote, P. J. et al. (1998), Antimicrob. Agents Chemother. 42, 2160-2170;Feder, R. et al. (2000), J. Biol. Chem. 275, 4230-4238; Gaidukov, L. etal. (2003), Biochemistry 42, 12866-12874; Kustanovich, I. et al. (2002),J. Biol. Chem. 277, 16941-16951; Mor, A. and Nicolas, P. (1994) J. Biol.Chem. 269, 1934-1939; Mor, A. et al. (1994), J. Biol. Chem. 269,31635-31641; Oh, D. et al. (2000), Biochemistry 39, 11855-11864;Patrzykat, A. et al. (2002), Antimicrob. Agents Chemother. 46, 605-614;Piers, K. L. and Hancock, R. E. (1994) Mol. Microbiol. 12, 951-958; andShepherd, C. M. et al. (2003), Biochemistry 370, 233-243).

The approaches described above have been applied in many studies aimingat designing novel AMPs. In these studies, the use of alpha-helix and/orbeta-sheet inducing building blocks, the use of the more flexiblebeta-amino acid building blocks, the use of mixed D- and L-amino acidsequences and the use of facially amphiphilic arylamide polymers, haveall demonstrated the importance of induced amphiphatic conformations onthe biological activity of AMPs.

Antimicrobial peptides can act in synergy with classical antibiotics,probably by enabling access of antibiotics into the bacterial cell(Darveau, R. P. et al. (1991), Antimicrob. Agents Chemother. 35,1153-1159; and Giacometti, A. et al. (2000), Diagn. Microbiol. Infect.Dis. 38, 115-118). Other potential uses include food preservation (Brul,S. and Coote, P. (1999), Int. J. Food Microbiol. 50, 1-17; Yaron, S.,Rydlo, T. et al. (2003), Peptides 24, 1815-1821; Appendini, P. andHotchkiss, J. H. (2000), J. Food Prot. 63, 889-893; and Johnsen, L. etal. (2000), Appl. Environ. Microbiol. 66, 4798-4802), imaging probes fordetection of bacterial or fungal infection loci (Welling, M. M. et al.(2000), Eur. J. Nucl. Med. 27, 292-301; Knight, L. C. (2003), Q. J.Nucl. Med. 47, 279-291; and Lupetti, A. et al. (2003), Lancet Infect.Dis. 3, 223-229), antitumor activity (Baker, M. A. et al. (1993), CancerRes. 53, 3052-3057; Jacob, L. and Zasloff, M. (1994), Ciba Found. Symp.186, 197-216; Johnstone, S. A. et al. (2000), Anticancer Drug Des 15,151-160; Moore, A. J. et al. (1994), Pept. Res. 7, 265-269; and Papo, N.and Shai, Y. (2003), Biochemistry 42, 9346-9354), mitogenic activity(Aarbiou, J. et al. (2002), J. Leukoc. Biol. 72, 167-174; Murphy, C. J.et al. (1993), J. Cell Physiol 155, 408-413; and Gudmundsson, G. H. andAgerberth, B. (1999), J. Immunol. Methods 232, 45-54) and lining ofmedical/surgical devices (Haynie, S. L. et al. (1995), Antimicrob.Agents Chemother. 39, 301-307).

However, while the potential of AMPs as new therapeutic agents is wellrecognized, the use of the presently known AMPs is limited by lack ofadequate specificity, and optional systemic toxicity (House of Lords,Science and Technology 7th Report: Resistance to antibiotics and otherantimicrobial agents. HL Paper 81-II, session, 1997-98; and Alan, A. R.et al. (2004), Plant Cell Rep. 22, 388-396). Thus, there is a clear needfor developing new antimicrobial peptides with improved specificity andtoxicity profile.

Moreover, although peptides are recognized as promising therapeutic andantimicrobial agents, their use is severely limited by their in vivo andex vivo instability and by poor pharmacokinetics. Peptides andpolypeptides are easily degraded in oxidative and acidic environmentsand therefore typically require intravenous administration (so as toavoid, e.g., degradation in the gastrointestinal tract). Peptides arefurther broken down in the blood system by proteolytic enzymes and arerapidly cleared from the circulation. Moreover, peptides are typicallycharacterized by poor absorption after oral ingestion, in particular dueto their relatively high molecular mass and/or the lack of specifictransport systems. Furthermore, peptides are characterized by highsolubility and therefore fail to cross biological barriers such as cellmembranes and the blood brain barrier, but exhibit rapid excretionthrough the liver and kidneys. The therapeutic effect of peptides isfurther limited by the high flexibility thereof, which counteracts theirreceptor-affinity due to the steep entropy decrease upon binding and aconsiderable thermodynamic energy cost. In addition, peptides are heatand humidity sensitive and therefore their maintenance requires costlycare, complex and inconvenient modes of administration, and high-cost ofproduction and maintenance. The above disadvantages impede the use ofpeptides and polypeptides as efficient drugs and stimulate the quest foran alternative, which oftentimes involves peptidomimetic compounds.

Peptidomimetic compounds are modified polypeptides which are designed tohave a superior stability, both in vivo and ex vivo, and yet at leastthe same receptor affinity, as compared with their parent peptides. Inorder to design efficacious peptidomimetics, an utmost detailedthree-dimensional understanding of the interaction with the intendedtarget is therefore required.

One method attempting at achieving the above goal utilizes syntheticcombinatorial libraries (SCLs), a known powerful tool for rapidlyobtaining optimized classes of active compounds. Thus, a number of novelantimicrobial compounds ranging from short peptides to smallheterocyclic molecules have been identified from SCLs (Blondelle, S. E.and Lohner, K. (2000), Biopolymers 55, 74-87).

Several families of naturally occurring modified peptides which exhibitstrong antimicrobial activity, have been uncovered in many organisms.These compounds, and their effective chemical alterations, have proposeda lead towards a general solution to the challenge of creating anantimicrobial compound devoid of the disadvantages associated withnatural AMPs.

Thus, for example, naturally occurring short antimicrobial peptidescharacterized by a lipophilic acyl chain at the N-terminus wereuncovered in various microorganisms (Bassarello, C. et al. (2004), J.Nat. Prod. 67, 811-816; Peggion, C., et al. (2003), J. Pept. Sci. 9,679-689; and Toniolo, C. et al. (2001), Cell Mol. Life Sci. 58,1179-1188). Acylation of AMPs was hence largely used as a technique toendow AMPs with improved antimicrobial characteristics (Avrahami, D. etal. (2001), Biochemistry 40, 12591-12603; Avrahami, D. and Shai, Y.(2002), Biochemistry 41, 2254-2263; Chicharro, C. et al. (2001),Antimicrob. Agents Chemother. 45, 2441-2449; Chu-Kung, A. F. et al.(2004), Bioconjug. Chem. 15, 530-535; Efron, L. et al. (2002), J. Biol.Chem. 277, 24067-24072; Lockwood, N. A. et al. (2004), Biochem. J. 378,93-103; Mak, P. et al. (2003), Int. J. Antimicrob. Agents 21, 13-19; andWakabayashi, H. et al. (1999), Antimicrob. Agents Chemother. 43,1267-1269). However, some studies indicate that attaching a hydrocarbonchain to the peptide, results in only marginal increase in the affinityof the lipopeptide to the membrane (Epand, R. M. (1997), Biopolymers 43,15-24).

One family of AMPs capable of alluding towards the main goal is thefamily of dermaseptins. Dermaseptins are peptides isolated from the skinof various tree frogs of the Phyllomedusa species (Brand, G. D. et al.(2002), J. Biol. Chem. 277, 49332-49340; Charpentier, S. et al. (1998),J. Biol. Chem. 273, 14690-14697; Mor, A. et al. (1991), Biochemistry 30,8824-8830; Mor, A. et al. (1994), Biochemistry 33, 6642-6650; Mor, A.and Nicolas, P. (1994), Eur. J. Biochem. 219, 145-154; andWechselberger, C. (1998), Biochim. Biophys. Acta 1388, 279-283). Theseare structurally and functionally related cationic peptides, typicallyhaving 24-34 amino acid residues. Dermaseptins were found to exert rapidcytolytic activity, from seconds to minutes, in vitro, against a varietyof microorganisms including viruses, bacteria, protozoa, yeast andfilamentous fungi (Coote, P. J. et al. (1998), Antimicrob. AgentsChemother. 42, 2160-2170; Mor, A. and Nicolas, P. (1994), J. Biol. Chem.269, 1934-1939; Mor, A. et al. (1994), J. Biol. Chem. 269, 31635-31641;Mor, A. and Nicolas, P. (1994), Eur. J. Biochem. 219, 145-154; Belaid,A. et al. (2002), J. Med. Virol. 66, 229-234; De Lucca, A. J. et al.(1998), Med. Mycol. 36, 291-298; Hernandez, C. et al. (1992), Eur. J.Cell Biol. 59, 414-424; and Mor, A. et al. (1991), J. Mycol. Med 1,5-10) as well as relatively inaccessible pathogens such as intracellularparasites (Efron, L. et al. (2002), J. Biol. Chem. 277, 24067-24072;Dagan, A. et al. (2002), Antimicrob. Agents Chemother. 46, 1059-1066;Ghosh, J. K. et al. (1997), J. Biol. Chem. 272, 31609-31616; andKrugliak, M. et al. (2000), Antimicrob. Agents Chemother. 44,2442-2451).

Since dermaseptins portray the biodiversity existing in a very largegroup of antimicrobial peptides in terms of structural and biologicalproperties, they serve as a general model system for understanding thefunction(s) of cationic antimicrobial peptides.

The 28-residue peptide dermaseptin S4 is known to bind avidly tobiological membranes and to exert rapid cytolytic activity against avariety of pathogens as well as against erythrocytes (Mor, A. et al.(1994), J. Biol. Chem. 269(50): 31635-41).

In a search for an active derivative (peptidomimetic) of S4, a28-residue derivative in which the amino acid residues at the fourth andtwentieth positions were replaced by lysine residues, known as K₄K₂₀-S4,and two short derivatives of 16 and 13 residues in which the amino acidresidue at the fourth position was replaced by a lysine residue, knownas K₄-S4(1-16) and K₄-S4(1-13), respectively, were prepared and testedfor the inhibitory effect thereof (Feder, R. et al. (2000), J. Biol.Chem. 275, 4230-4238). The minimal inhibitory concentrations (MICs) ofthese derivatives for 90% of the 66 clinical isolates tested (i.e.,MIC₉₀ for S. aureus, P. aeruginosa and E. coli), varied between 2 and 8μg/ml for the various species, whereby the 13-mer derivative K₄-S4(1-13)was found to be significantly less hemolytic when incubated with humanerythrocytes, as compared with similarly active derivatives of magaininand protegrin, two confirmed antimicrobial peptide families (Fahrner, R.L. et al. (1996), Chem. Biol. 3(7): 543-50; Zasloff, M. et al. (1988),Proc. Natl. Acad. Sci. USA 85(3): 910-3; Yang L. et al. (2000), Biophys.J., 79 2002-2009). Additional studies further confirmed that short,lysine-enriched S4 derivatives, are promising anti-microbial agents bybeing characterized by reduced toxicity and by showing efficacy alsoafter pre-exposure of the subjects thereto.

N-terminal acylation of the C-terminally truncated 13-mer S4 derivativeK₄-S4(1-13) also resulted in reduced hemolytic activity, whereby severalderivatives, such as its aminoheptanoyl derivative, displayed potent andselective activity against the intracellular parasite, i.e., increasedantiparasitic efficiency and reduced hemolysis. These studies indicatethat increasing the hydrophobicity of anti-microbial peptides enhancetheir specificity, presumably by allowing such AMPs to act specificallyon the membrane of intracellular parasites and thus support a proposedmechanism according to which the lipopeptide crosses the host cellplasma membrane and selectively disrupts the parasite membrane(s).

Overall, the data collected from in-vitro and in-vivo experimentsindicated that some dermaseptin derivatives could be useful in thetreatment of a variety of microbial-associated conditions includinginfections caused by multidrug-resistant pathogens. These agents werefound highly efficacious, and no resistance was appeared to develop upontheir administration. Nevertheless, the therapeutic use of these agentsis still limited by the in vivo and ex vivo instability thereof, by poorpharmacokinetics, and by other disadvantageous characteristics ofpeptides, as discussed hereinabove.

In conclusion, most of the presently known antimicrobial peptides andpeptidomimetics are of limited utility as therapeutic agents despitetheir promising antimicrobial activity. The need for compounds whichhave AMP characteristics, and are devoid of the limitations associatedwith AMPs is still present, and the concept of providing chemically andmetabolically-stable active compounds in order to achieve enhancedspecificity and hence enhanced clinical selectivity has been widelyrecognized.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, novel, metabolically-stable, non-toxic andcost-effective antimicrobial agents devoid of the above limitations.

SUMMARY OF THE INVENTION

The present inventors have now designed and successfully prepared anovel class of polymeric compounds, which are based on positivelycharged amino acid residues and hydrophobic moieties. These novelpolymers were found highly efficient as selective antimicrobial agents,while being devoid of toxicity and resistance induction.

Thus, according to one aspect of the present invention there is provideda polymer which includes two or more amino acid residues and one or morehydrophobic moiety residues, wherein one or more of the hydrophobicmoiety residues is being covalently linked to at least two amino acidresidues via the N-alpha of one amino acid residue and via the C-alphaof another amino acid residue.

According to further features in preferred embodiments of the inventiondescribed below, the polymer is having an antimicrobial activity.

According to still further features in the described preferredembodiments the polymer is capable of selectively destructing at least aportion of the cells of a pathogenic microorganism.

According to still further features in the described preferredembodiments the pathogenic microorganism is selected from the groupconsisting of a prokaryotic organism, an eubacterium, anarchaebacterium, a eukaryotic organism, a yeast, a fungus, an alga, aprotozon and a parasite.

According to still further features in the described preferredembodiments the polymer includes at least two hydrophobic moietyresidues, wherein one or more of the hydrophobic moiety residues arelinked to the N-alpha of an amino acid residue at the N-terminus of oneof the amino acid residues and/or the C-alpha of another amino acidresidue at the C-terminus.

According to still further features in the described preferredembodiments the polymer includes two or more hydrophobic moietyresidues, wherein one or more of the hydrophobic moiety residues arelinked to the side-chain of an amino acid residue in the polymer.

According to still further features in the described preferredembodiments one or more of the amino acid residues is a positivelycharged amino acid residue.

According to still further features in the described preferredembodiments the positively charged amino acid residue is selected fromthe group consisting of a histidine residue, a lysine residue, anornithine residue and an arginine residue.

According to yet further features of the present invention, one or moreof the hydrophobic moiety residues is linked to one or more of the aminoacid residues via a peptide bond.

According to still further features in the described preferredembodiments one or more of the hydrophobic moiety residues is linked totwo amino acid residues via a peptide bond

According to still further features in the described preferredembodiments one or more of the hydrophobic moiety residues is linked toeach of the amino acid residues via a peptide bond.

According to still further features in the described preferredembodiments one or more of the hydrophobic moiety residues is linked tothe N-alpha of the amino acid residue via a peptide bond.

According to still further features in the described preferredembodiments one or more of the hydrophobic moiety residues is linked tothe C-alpha of the amino acid residue via a peptide bond.

According to still further features in the described preferredembodiments one or more of the hydrophobic moieties has a carboxylicgroup at one end thereof and an amine group at the other end thereof.

According to still further features in the described preferredembodiments the polymer includes from 2 to 50 amino acid residues,preferably from 2 to 12 amino acid residues and more preferably from 2to 8 amino acid residues.

According to still further features in the described preferredembodiments the polymer includes from 1 to 50 hydrophobic moietyresidues, preferably from 1 to 12 hydrophobic moiety residues and morepreferably from 1 to 8 hydrophobic moiety residues.

According to still further features in the described preferredembodiments the hydrophobic moiety residue includes one or morehydrocarbon chains which have from 4 to 30 carbon atoms.

According to still further features in the described preferredembodiments the hydrophobic moiety residue includes one or more fattyacid residues which are selected from the group consisting of anunbranched saturated fatty acid residue, a branched saturated fatty acidresidue, an unbranched unsaturated fatty acid residue, a branchedunsaturated fatty acid residue and any combination thereof, and thefatty acid residue has from 4 to 30 carbon atoms.

According to still further features in the described preferredembodiments the fatty acid residue is selected from the group consistingof a butyric acid residue, a caprylic acid residue and a lauric acidresidue.

According to still further features in the described preferredembodiments one or more of the hydrophobic moieties is an ω-amino-fattyacid residue. The ω-amino-fatty acid residue is selected from the groupconsisting of 4-amino-butyric acid, 6-amino-caproic acid,8-amino-caprylic acid, 10-amino-capric acid, 12-amino-lauric acid,14-amino-myristic acid, 16-amino-palmitic acid, 18-amino-stearic acid,18-amino-oleic acid, 16-amino-palmitoleic acid, 18-amino-linoleic acid,18-amino-linolenic acid and 20-amino-arachidonic acid. Preferably, theω-amino-fatty acid residue is selected from the group consisting of4-amino-butyric acid, 8-amino-caprylic acid and 12-amino-lauric acid.

According to still further features in the described preferredembodiments all of the amino acid residues of the polymer are positivelycharged amino acid residues, such as lysine residues, histidineresidues, ornithine residues, arginine residues and any combinationsthereof.

According to still further features in the described preferredembodiments all the positively charged amino acid residues are lysineresidues.

According to still further features of the preferred embodiments of theinvention described below, the polymer further includes one or moreactive agent attached thereto.

According to still further features in the described preferredembodiments the active agent is attached to a side chain of an aminoacid residue, either via the N-alpha of the amino acid residue at theN-terminus and/or the C-alpha of the amino acid residue at theC-terminus, and/or to one or more of the hydrophobic moiety residues ofthe polymer.

According to still further features in the described preferredembodiments the active agent is a labeling agent, which is selected fromthe group consisting of a fluorescent agent, a radioactive agent, amagnetic agent, a chromophore, a phosphorescent agent and a heavy metalcluster.

According to still further features in the described preferredembodiments the active agent comprises at least one therapeuticallyactive agent, which is selected from the group consisting of an agonistresidue, an amino acid residue, an analgesic residue, an antagonistresidue, an antibiotic agent residue, an antibody residue, anantidepressant agent, an antigen residue, an anti-histamine residue, ananti-hypertensive agent, an anti-inflammatory drug residue, ananti-metabolic agent residue, an antimicrobial agent residue, anantioxidant residue, an anti-proliferative drug residue, an antisenseresidue, a chemotherapeutic drug residue, a co-factor residue, acytokine residue, a drug residue, an enzyme residue, a growth factorresidue, a heparin residue, a hormone residue, an immunoglobulinresidue, an inhibitor residue, a ligand residue, a nucleic acid residue,an oligonucleotide residue, a peptide residue, a phospholipid residue, aprostaglandin residue, a protein residue, a toxin residue, a vitaminresidue and any combination thereof.

According to still further features in the described preferredembodiments the polymer is capable of delivering one or more activeagents, such as a labeling agent or a therapeutically active agent, toat least a portion of the cells of a pathogenic microorganism asdescribed herein.

According to still further features in the described preferredembodiments the polymers are selected from the compounds presented inTable 3 hereinbelow.

According to still further features in the described preferredembodiments the polymer described herein can be represented by thegeneral formula I:

X-W₀-[A₁-Z₁-D₁]-W₁-[A₂-Z₂-D₂]-W₂- . . . [An-Zn-Dn]-Wn-Y  Formula I

wherein:

n is an integer from 2 to 50, preferably from 2 to 12 and morepreferably from 2 to 8;

A₁, A₂, . . . , An are each independently an amino acid residue,preferably a positively charged amino acid residue, and more preferablyall of A₁, A₂, . . . , An are positively charged amino acid residues asdiscussed hereinabove, such as histidine residues, lysine residues,ornithine residues and arginine residues;

D₁, D₂, . . . , Dn are each independently a hydrophobic moiety residue,as described herein, or absent, provided that at least one suchhydrophobic moiety residue exists in the polymer, and preferably atleast one of the hydrophobic moiety residues is a ω-amino-fatty acidresidue;

Z₁, Z₂, . . . , Zn and W₀, W₁, W₂, . . . , Wn are each independently alinking moiety linking an amino acid residue and a hydrophobic moietyresidue or absent, preferably at least one of the linking moieties is apeptide bond and most preferable all the linking moieties are peptidebonds;

X and Y may each independently be hydrogen, an amine, an amino acidresidue, a hydrophobic moiety residue, another polymer having thegeneral Formula I or absent.

According to still further features in the described preferredembodiments the polymer further includes one or more active agent, asdescribed herein, attached to one or more of either X, Y, W₀, A₁, Anand/or Wn.

According to another aspect of the present invention there is provided aconjugate which includes an amino acid residue and a hydrophobic moietyresidue attached to the N-alpha or the C-alpha of the amino acidresidue, the hydrophobic moiety residue being designed capable offorming a bond with an N-alpha or a C-alpha of an additional amino acidresidue.

According to further features in the preferred embodiments of theinvention described below, the hydrophobic moiety residue is attached tothe N-alpha or the C-alpha of the amino acid residue via a peptide bond.

According to still further features in the described preferredembodiments the hydrophobic moiety has a carboxylic group at one endthereof and an amine group at the other end thereof and further includesa hydrocarbon chain as described herein.

According to still further features in the described preferredembodiments the hydrophobic moiety includes a fatty acid residue asdescribed herein.

According to still further features in the described preferredembodiments the hydrophobic moiety is an ω-amino-fatty acid residue asdescribed herein.

According to still another aspect of the present invention there isprovided a process of preparing the conjugate described hereinabove, theprocess comprises providing an amino acid; providing a hydrophobicmoiety having a first functional group that is capable of reacting withan N-alpha of an amino acid residue and/or a second functional groupcapable of reacting with a C-alpha of an amino acid; linking the firstfunctional group in the hydrophobic moiety to the amino acid via theN-alpha of said amino acid; or linking the second functional group inthe hydrophobic moiety to the amino acid via the C-alpha of the aminoacid. Preferably the hydrophobic moiety is linked to the amino acid viaa peptide bond.

According to further features in the preferred embodiments of theinvention described below, the amino acid is a positively charged aminoacid such as, for example, histidine, lysine, ornithine and arginine.

According to further features in the preferred embodiments all thepositively charged amino acids are lysines.

According to still further features in the described preferredembodiments the hydrophobic moiety has a carboxylic group at one endthereof, an amine group at the other end thereof and a hydrocarbonchain, as described herein.

According to still further features in the described preferredembodiments the hydrophobic moiety includes a fatty acid residue asdescribed herein.

According to still further features in the described preferredembodiments the hydrophobic moiety is an ω-amino-fatty acid residue asdescribed herein.

According to still further features in the described preferredembodiments the hydrophobic moiety is a 8-amino-caprylic acid.

According to still further features in the described preferredembodiments, n is an integer from 6 to 8.

According to still further features in the described preferredembodiments, the polymer has the formula (SEQ ID NO: 45):

According to still further features in the described preferredembodiments, the polymer has the formula (SEQ ID NO: 43):

According to yet another aspect of the present invention there isprovided a pharmaceutical composition which includes as an activeingredient the polymer of the present invention, described herein, and apharmaceutically acceptable carrier.

According to further features in the preferred embodiments of theinvention described below, the pharmaceutical composition is packaged ina packaging material and identified in print, in or on said packagingmaterial, for use in the treatment of a medical condition associatedwith a pathogenic microorganism such as a prokaryotic organism, aneubacterium, an archaebacterium, a eukaryotic organism, a yeast, afungus, an alga, a protozon and a parasite.

According to still further features in the described preferredembodiments the pharmaceutical composition further includes one or moreadditional therapeutically active agent as described herein, wherebypreferably the therapeutically active agent includes an antibioticagent.

According to another aspect of the present invention there is provided amethod of treating a medical condition associated with a pathogenicmicroorganism, as described herein, the method includes administering toa subject in need thereof a therapeutically effective amount of thepolymer described herein.

According to further features in the preferred embodiments of theinvention described below, the administration is effected orally,rectally, intravenously, topically, intranasally, intradermally,transdermally, subcutaneously, intramuscularly, intrperitoneally or byintrathecal catheter.

According to still further features in the described preferredembodiments the method further includes administering to the subject oneor more therapeutically active agent as described herein, preferably, anantibiotic agent.

According to still further features in the described preferredembodiments the polymer of the present invention is administered eitherper se or as a part of a pharmaceutical composition; the pharmaceuticalcomposition further includes a pharmaceutically acceptable carrier, asdescribed herein.

According to an additional aspect of the present invention there isprovided a medical device which includes the polymer of the presentinvention and a delivery system configured for delivering the polymer toa bodily site of a subject.

According to further features in the preferred embodiments of theinvention described below, the polymer forms a part of a pharmaceuticalcomposition, and the pharmaceutical composition further includes apharmaceutically acceptable carrier.

According to still further features in the described preferredembodiments the delivery is effected by inhalation, and the deliverysystem is selected from the group consisting of a metered dose inhaler,a respirator, a nebulizer inhaler, a dry powder inhaler, an electricwarmer, a vaporizer, an atomizer and an aerosol generator.

According to still further features in the described preferredembodiments the delivery is effected transdermally, and the deliverysystem is selected from the group consisting of an adhesive plaster anda skin patch.

According to still further features in the described preferredembodiments the delivery is effected topically and the delivery systemis selected from the group consisting of an adhesive strip, a bandage,an adhesive plaster, a wound dressing and a skin patch.

According to still further features in the described preferredembodiments the delivery is effected by implanting the medical device ina bodily organ. Preferably the delivery system further includes abiocompatible matrix which in turn includes a biodegradable polymer andfurther includes a slow release carrier.

According to still an additional aspect of the present invention thereis provided a food preservative which includes an effective amount ofthe polymer of the present invention, and preferably further includes anedible carrier.

According to a further aspect of the present invention there is providedan imaging probe for detecting a pathogenic microorganism as describedherein, which includes a polymer as described herein, and one or morelabeling agent, as described herein, attached thereto.

According to further features in the preferred embodiments of theinvention described below, the labeling agent(s) is attached to a sidechain of an amino acid residue, a C-terminus and/or a N-terminus of thepolymer and/or one of the hydrophobic residues of the polymer of thepresent invention.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a novel class ofantimicrobial polymers, which combine the merits of therapeuticallyactive antimicrobial peptides, e.g., high efficacy and specificity,without exhibiting the disadvantages of peptides.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 presents a cumulative bar graph demonstrating the highcorrelation between the antimicrobial activity and the hydrophobicity ofexemplary polymers according to the present invention, by marking thepolymers which exhibited a significant microbial activity (MIC value ofless than 50 μM) against E. coli (in red bars), P. aeruginosa (in yellowbars), methicilin-resistant S. aureus (in blue bars) and B. cereus (ingreen bars), on the scale of the acetonitrile percentages in the mobilephase at which the polymers were eluted on a reverse phase HPLC column;

FIG. 2 presents a cumulative bar graph demonstrating the lack ofcorrelation between the antimicrobial activity and the net positivecharge of exemplary polymers according to the present invention, bymarking the polymers which exhibited a significant microbial activity(MIC value of less than 50 μM) against E. coli (in red bars), P.aeruginosa (in yellow bars), methicilin-resistant S. aureus (in bluebars) and B. cereus (in green bars), over bins representing the netpositive charge from +9 to +1;

FIGS. 3A-3C presents a bar graph demonstrating the non-resistanceinducing effect of exemplary polymers according to the presentinvention, by measuring MICs level evolution on E. coli after 10iterations of successive exposures of bacteria to sub-lyticconcentrations of K(NC₁₂K)₃NH₂ (SEQ ID NO: 60) and C₁₂K(NC₈K)₅NH₂ (SEQID NO: 43), as compared to exposures to three classical antibioticagents, tetracycline, gentamycin and ciprofloxacin (FIG. 3a ), and onmethicilin-resistant S. aureus after 15 iterations of successiveexposures of bacteria to sub-lytic concentrations of C₁₂KKNC₁₂KNH₂ (SEQID NO: 78), as compared to exposures to two antibiotic agents,rifampicin and tetracycline (FIG. 3b ), and the development ofresistance of E. coli to C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), evaluatedduring 15 serial passages, as compared to exposures to three classicalantibiotic agents, ciprofloxacin, imipenem, and tetracycline (FIG. 3c )(the relative MIC is the normalized ratio of the MIC obtained for agiven subculture to the concomitantly determined MIC obtained onbacteria harvested from control wells (wells cultured withoutantimicrobial agent) from the previous generation;

FIG. 4 presents comparative plots demonstrating the kinetic bactericidaleffect of C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43), an exemplary polymer accordingto the present invention, on E. coli. incubated in the presence of thepolymer, with colony forming units (CFU) counts performed after thespecified incubation periods and compared in a dose-dependent experimentat zero (control), 3 and 6 multiples of the minimal inhibitoryconcentration (MIC) value (3.1 μM) in LB medium at 37° C.;

FIG. 5 presents comparative plots demonstrating the kinetic bactericidaleffect of C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplary polymer accordingto the present invention (black triangles), on E. coli., compared withnormal bacterial growth control (black circles), and with kineticbactericidal effect of while Imipenem (white squares) and Ciprofloxacin(black squares), as determined at a concentration corresponding to sixmultiples of their respective MIC value (plotted values represent themean±standard deviations obtained from at least two independentexperiments);

FIG. 6 presents comparative plots demonstrating the hemolytic effect ofC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplary polymer according to thepresent invention, compared with the hemolytic effect of bivalirudin, asynthetic peptide and FDA approved thrombin inhibitor, and with thehemolytic effect of MSI-78, a magainin derivative, determined againsthuman RBC (10% hematocrit) after 1 hour incubation at 37° C. in thepresence of 31 μM (striped bars), 94 μM (gray bars) and 156 μM (whitebars) polymer/peptide concentration (plotted values represent themean±standard deviations obtained from at least four independentexperiments);

FIG. 7 presents the circular dichroism spectra of two exemplary polymersaccording to the present invention, C₁₂K(NC₈K)₅NH₂ and C₁₂K(NC₈K)₇NH₂(SEQ ID NOs: 43 and 45, respectively), taken in the designated media atpolymer concentration of 100 μM (liposome concentration of 2 mM),expressed as mean residue molar ellipticity, and compared with a15-residue control peptide, an acylated dermaseptin S4 derivative (datarepresent average values from three separate recordings);

FIG. 8 presents the circular dichroism spectra of C₁₂K(NC₈K)₇NH₂ (SEQ IDNO: 45), another exemplary polymer according to the present invention(gray lines), and of a control antimicrobial peptide K₄S₄(1-16) (blacklines) (SEQ ID NO: 98), taken in PBS alone (dashed lines) or in thepresence of 2 mM POPC:POPG (3:1) liposomes concentration suspended inPBS (solid lines) (data represent average values from three separaterecordings);

FIG. 9 presents association and dissociation curves (binding rates)obtained by surface plasmon resonance (SPR) measurements, demonstratingthe membrane binding properties of various doses (0.21, 0.42, 0.84,1.67, and 3.35 μg) of C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43), an exemplarypolymer according to the present invention, to a model membrane (K_(app)is the resulting binding constants calculated assuming a 2-step model);

FIG. 10 presents a bar graph demonstrating the binding of exemplarypolymers according to the present invention, denoted as KNC₈KNH₂,K(NC₈K)₂NH₂, K(NC₈K)₃NH₂, K(NC₈K)₆NH₂, KNC₁₂KNH₂, K(NC₁₂K)₂NH₂ andK(NC₁₂K)₃NH₂ (SEQ ID NOs: 32, 33, 34, 37, 58, 59 and 60, respectively),to lipopolysaccharide, as measured by SPR, wherein the weaker binding ofthe polymers to liposomes after incubation with LPS substantiates thatthe polymers are bound to the LPS;

FIG. 11 presents a photograph of a UV illuminated 1% agarose gelelectrophoresis, demonstrating the DNA binding characteristics ofC₁₂KKNC₁₂KNH₂, K(NC₄K)₇NH₂ and C₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 78, 14 and60, respectively), exemplary polymers according to the presentinvention, as measured by DNA retardation assay after the polymers wereincubated for 30 minutes at room temperature at the specifiedDNA/polymer ratios (w:w) using 200 nanograms of plasmid (normalmigration in absence of the polymer of the plasmid pUC19 is shown inleftmost lane);

FIG. 12 presents comparative plots demonstrating the antimicrobialactivity of C₈K(NC₈K)₇NH₂ (SEQ ID NO: 31), an exemplary polymeraccording to the present invention (in black circles), against themicro-flora found in human saliva, as compared to IB-367 (SEQ ID NO:97), a peptide with known antimicrobial activity (in white circles) andthe vehicle buffer as control (white triangle) in logarithmic units ofCFU per ml versus incubation time;

FIG. 13 presents a comparative plot demonstrating the anti-malarialactivity of C₁₂K(NC₁₂K)₃NH₂ (SEQ ID NO: 56), an exemplary polymeraccording to the present invention, by showing the effect of time ofexposure of the malaria causing parasites to the polymer on thestage-dependent effect on Plasmodium falciparum parasite viability(chloroquine-resistant FCR3 strain versus chloroquine-sensitive NF54strain);

FIG. 14 presents a comparative plot demonstrating the anti-malarialactivity of C₁₂KNC₈KNH₂ (SEQ ID NO: 39), an exemplary polymer accordingto the present invention, by showing the effect of time of treatment atdifferent parasite developmental stages with the polymer, on parasiteviability;

FIGS. 15A-15B present the rate of survival, monitored over time periodof 7 days, of infected mice (n=10 per group) inoculatedintraperitoneally with 2.5×10⁶ CFUs of E. coli CI 3504 (FIG. 15a ) and5×10⁶ CFUs of E. coli (FIG. 15b ), and subsequently treatedintraperitoneally with PBS (black circles), with a single dose of 4mg/kg C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) (gray squares) or with four dosesof 2 mg/kg Imipenem (asterisk), demonstrating the high in-vivo efficacyof the polymers of the present invention; and

FIG. 16 presents the rate of survival, monitored over time period of 6days, of mice (n=12 per group) treated intraperitoneally with a blankcontrol (white bars), 4 mg/kg body weight (sparsely striped bars), 10mg/kg body weight (densely striped bars) and 20 mg/kg body weight (blackbars) of C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 39), demonstrating the low toxicityof the polymers of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a novel class of polymeric antimicrobialagents, which are designed to exert antimicrobial activity while beingstable, non-toxic and avoiding development of resistance thereto, andcan therefore be beneficially utilized in the treatment of variousmedical conditions associated with pathogenic microorganisms. Thepresent invention is further of pharmaceutical compositions, medicaldevices and food preservatives containing same. The antimicrobialpolymers of the present invention preferably include one or morepositively charged amino acid residues and one or hydrophobic moietyresidues attached one to another.

The principles and operation of the present invention may be betterunderstood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As discussed above, the use of classical modern antibiotic agents suchas tetracycline, gentamycin, ciprofloxacin and methicillin has becomeduring the years severely limited by the development of resistancethereto. Extensive studies have therefore been conducted in a search fornovel antimicrobial agents that would circumvent the resistanceinduction.

As further discussed above, naturally occurring antimicrobial peptides(AMPs) are exceptionally potent antimicrobial agents, but aspharmaceuticals they suffer from the limitations associated with peptideproduction, maintenance and modes of clinical administration fortherapeutic use.

Based on the knowledge which accumulated over the years on the nature ofantimicrobial peptides and the limitations associated with their use,the present inventors hypothesized that in order to achieve a novelclass of antimicrobial agents devoid of the resistance-inducingdrawbacks of classical antibiotic agents, and those of AMPs, three keyattributes of AMPs needs to be maintained: a flexible structure, anamphiphatic character and a net positive charge.

While conceiving the present invention, it was envisioned that aflexible polymeric structure will serve the objective of avoiding thedevelopment of resistance in the target microorganism. It was furtherenvisioned that use of amino acids, as defined hereinbelow, can serve asa basis for both a polymer as well as a source for net positive charge.

While further conceiving the present invention, it was hypothesized thatavoiding a pure amino acid polypeptide structure would not only resolvethe production and maintenance issues limiting the use of polypeptidesas drugs, but would also alleviate the sever limitations restricting theadministration of polypeptides as drugs. Thus, it was envisioned thatthe desired amphiphatic trait of the envisioned polymer may arise fromnon-amino acid hydrophobic moieties, such as, but not limited to fattyacids and the likes.

While reducing the present invention to practice, as is demonstrated inthe Examples section that follows, the present inventors have developedand successfully produced a novel class of polymers which were shown toexhibit high antimicrobial activity, low resistance induction,non-hemolyticity, resistibility to plasma proteases and high affinity tomicrobial membranes.

While further conceiving the present invention, it was envisioned thatconjugating an active agent to the polymeric structure, such as alabeling agent and/or a therapeutically active agent, will combine theaffinity of the polymers of the present invention to microbial cells,and the utility of the additional active agent. In cases where theactive agent is a labeling agent, the combination will assist inlocating and diagnosing concentration of microbial growth in a host, andin cases where the active agent is a therapeutically active agent,synergistic therapeutic effects could be achieved, resulting from thedual therapeutic effect of the therapeutically active agent and theantimicrobial polymeric structure. In addition, targeted delivery of thetherapeutic agent could be achieved.

Thus, according to one aspect of the present invention, there isprovided a polymer, having an antimicrobial activity, which comprises aplurality (e.g., two or more) amino acid residues and one or morehydrophobic moiety residues, wherein at least one of the hydrophobicmoiety residues is covalently linked to at least two amino acid residuesvia the N-alpha of one amino acid residue and/or the C-alpha of theother amino acid residue. Therefore, the polymer is a chain made of asequence of amino acid residues, interrupted by one or more hydrophobicmoiety residues.

As used herein throughout the term “amino acid” or “amino acids” isunderstood to include the 20 genetically coded amino acids; those aminoacids often modified post-translationally in vivo, including, forexample, hydroxyproline, phosphoserine and phosphothreonine; and otherunusual amino acids including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.Furthermore, the term “amino acid” includes both D- and L-amino acidsand other non-naturally occurring amino acids.

Tables 1 and 2 below list the genetically encoded amino acids (Table 1)and non-limiting examples of non-conventional/modified amino acids(Table 2) which can be used with the present invention.

TABLE 1 Amino acid Three-Letter Abbreviation One-letter Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Iie I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

TABLE 2 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane-carboxylate Cpro L-N-methylasparagine Nmasnaminoisobutyric acid Aib L-N-methylaspartic acid Nmaspaminonorbornyl-carboxylate Norb L-N-methylcysteine NmcysCyclohexylalanine Chexa L-N-methylglutamine Nmgin CyclopentylalanineCpen L-N-methylglutamic acid Nmglu D-alanine Dal L-N-methylhistidineNmhis D-arginine Darg L-N-methylisolleucine Nmile D-aspartic acid DaspL-N-methylleucine Nmleu D-cysteine Dcys L-N-methyllysine NmlysD-glutamine Dgln L-N-methylmethionine Nmmet D-glutamic acid DgluL-N-methylnorleucine Nmnle D-histidine Dhis L-N-methylnorvaline NmnvaD-isoleucine Dile L-N-methylornithine Nmorn D-leucine DleuL-N-methylphenylalanine Nmphe D-lysine Dlys L-N-methylproline NmproD-methionine Dmet L-N-methylserine Nmser D/L-ornithine D/LornL-N-methylthreonine Nmthr D-phenylalanine Dphe L-N-methyltryptophanNmtrp D-proline Dpro L-N-methyltyrosine Nmtyr D-serine DserL-N-methylvaline Nmval D-threonine Dthr L-N-methylethylglycine NmetgD-tryptophan Dtrp L-N-methyl-t-butylglycine Nmtbug D-tyrosine DtyrL-norleucine Nle D-valine Dval L-norvaline Nva D-α-methylalanine Dmalaα-methyl-aminoisobutyrate Maib D-α-methylarginine Dmargα-methyl-γ-aminobutyrate Mgabu D-α-methylasparagine Dmasnα-methylcyclohexylalanine Mchexa D-α-methylaspartate Dmaspα-methylcyclopentylalanine Mcpen D-α-methylcysteine Dmcysα-methyl-α-napthylalanine Manap D-α-methylglutamine Dmglnα-methylpenicillamine Mpen D-α-methylhistidine DmhisN-(4-aminobutyl)glycine Nglu D-α-methylisoleucine DmileN-(2-aminoethyl)glycine Naeg D-α-methylleucine DmleuN-(3-aminopropyl)glycine Norn D-α-methyllysine DmlysN-amino-α-methylbutyrate Nmaabu D-α-methylmethionine Dmmetα-napthylalanine Anap D-α-methylornithine Dmorn N-benzylglycine NpheD-α-methylphenylalanine Dmphe N-(2-carbamylethyl)glycine NglnD-α-methylproline Dmpro N-(carbamylmethyl)glycine Nasn D-α-methylserineDmser N-(2-carboxyethyl)glycine Nglu D-α-methylthreonine DmthrN-(carboxymethyl)glycine Nasp D-α-methyltryptophan DmtrpN-cyclobutylglycine Ncbut D-α-methyltyrosine Dmty N-cycloheptylglycineNchep D-α-methylvaline Dmval N-cyclohexylglycine Nchex D-α-methylalnineDnmala N-cyclodecylglycine Ncdec D-α-methylarginine DnmargN-cyclododeclglycine Ncdod D-α-methylasparagine DnmasnN-cyclooctylglycine Ncoct D-α-methylasparatate DnmaspN-cyclopropylglycine Ncpro D-α-methylcysteine DnmcysN-cycloundecylglycine Ncund D-N-methylleucine DnmleuN-(2,2-diphenylethyl)glycine Nbhm D-N-methyllysine DnmlysN-(3,3-diphenylpropyl)glycine Nbhe N-methylcyclohexylalanine NmchexaN-(3-indolylyethyl) glycine Nhtrp D-N-methylornithine DnmornN-methyl-γ-aminobutyrate Nmgabu N-methylglycine NalaD-N-methylmethionine Dnmmet N-methylaminoisobutyrate NmaibN-methylcyclopentylalanine Nmcpen N-(1-methylpropyl)glycine NileD-N-methylphenylalanine Dnmphe N-(2-methylpropyl)glycine NileD-N-methylproline Dnmpro N-(2-methylpropyl)glycine Nleu D-N-methylserineDnmser D-N-methyltryptophan Dnmtrp D-N-methylserine DnmserD-N-methyltyrosine Dnmtyr D-N-methylthreonine Dnmthr D-N-methylvalineDnmval N-(1-methylethyl)glycine Nva γ-aminobutyric acid GabuN-methyla-napthylalanine Nmanap L-t-butylglycine TbugN-methylpenicillamine Nmpen L-ethylglycine EtgN-(p-hydroxyphenyl)glycine Nhtyr L-homophenylalanine HpheN-(thiomethyl)glycine Ncys L-α-methylarginine Marg penicillamine PenL-α-methylaspartate Masp L-α-methylalanine Mala L-α-methylcysteine McysL-α-methylasparagine Masn L-α-methylglutamine MglnL-α-methyl-t-butylglycine Mtbug L-α-methylhistidine MhisL-methylethylglycine Metg L-α-methylisoleucine Mile L-α-methylglutamateMglu D-N-methylglutamine Dnmgln L-α-methylhomo phenylalanine MhpheD-N-methylglutamate Dnmglu N-(2-methylthioethyl)glycine NmetD-N-methylhistidine Dnmhis N-(3-guanidinopropyl)glycine NargD-N-methylisoleucine Dnmile N-(1-hydroxyethyl)glycine NthrD-N-methylleucine Dnmleu N-(hydroxyethyl)glycine Nser D-N-methyllysineDnmlys N-(imidazolylethyl)glycine Nhis N-methylcyclohexylalanine NmchexaN-(3-indolylyethyl)glycine Nhtrp D-N-methylornithine DnmornN-methyl-γ-aminobutyrate Nmgabu N-methylglycine NalaD-N-methylmethionine Dnmmet N-methylaminoisobutyrate NmaibN-methylcyclopentylalanine Nmcpen N-(1-methylpropyl)glycine NileD-N-methylphenylalanine Dnmphe N-(2-methylpropyl)glycine NleuD-N-methylproline Dnmpro D-N-methyltryptophan Dnmtrp D-N-methylserineDnmser D-N-methyltyrosine Dnmtyr D-N-methylthreonine DnmthrD-N-methylvaline Dnmval N-(1-methylethyl)glycine Nval γ-aminobutyricacid Gabu N-methyla-napthylalanine Nmanap L-t-butylglycine TbugN-methylpenicillamine Nmpen L-ethylglycine EtgN-(p-hydroxyphenyl)glycine Nhtyr L-homophenylalanine HpheN-(thiomethyl)glycine Ncys L-α-methylarginine Marg penicillamine PenL-α-methylaspartate Masp L-α-methylalanine Mala L-α-methylcysteine McysL-α-methylasparagine Masn L-α-methylglutamine MglnL-α-methyl-t-butylglycine Mtbug L-α-methylhistidine MhisL-methylethylglycine Metg L-α-methylisoleucine Mile L-α-methylglutamateMglu L-α-methylleucine Mleu L-α-methylhomophenylalanine MhpheL-α-methylmethionine Mmet N-(2-methylthioethyl)glycine NmetL-α-methylnorvaline Mnva L-α-methyllysine Mlys L-α-methylphenylalanineMphe L-α-methylnorleucine Mnle L-α-methylserine mser L-α-methylornithineMorn L-α-methylvaline Mtrp L-α-methylproline Mpro L-α-methylleucine MvalNnbhm L-α-methylthreonine MthrN-(N-(2,2-diphenylethyl)carbamylmethyl-glycine Nnbhm L-α-methyltyrosineMtyr 1-carboxy-1-(2,2-diphenyl ethylamino)cyclopropane NmbcL-N-methylhomophenylalanine NmhpheN-(N-(3,3-diphenylpropyl)carbamylmethyl(1)glycine Nnbhe D/L-citrullineD/Lctr

As used herein, the phrase “hydrophobic moiety” describes a chemicalmoiety that has a minor or no affinity to water, that is, which has alow or no dissolvability in water and often in other polar solvents.Exemplary suitable hydrophobic moieties for use in the context of thepresent invention, include, without limitation, hydrophobic moietiesthat consist predominantly of one or more hydrocarbon chains and/oraromatic rings, and one or more functional groups which may benon-hydrophobic, but do not alter the overall hydrophobicity of thehydrophobic moiety. Representative examples include, without limitation,fatty acids, hydrophobic amino acids (amino acids with hydrophobicside-chains), alkanes, alkenes, aryls and the likes, as these terms aredefined herein, and any combination thereof.

As used herein, the phrase “chemical moiety” describes a residue of achemical compound, which typically has certain functionality. As is wellaccepted in the art, the term “residue” refers herein to a major portionof a molecule which is covalently linked to another molecule.

As used herein, the phrase “functional group” describes a chemical groupthat is capable of undergoing a chemical reaction that typically leadsto a bond formation. The bond, according to the present invention, ispreferably a covalent bond. Chemical reactions that lead to a bondformation include, for example, nucleophilic and electrophilicsubstitutions, nucleophilic and electrophilic addition reactions,addition-elimination reactions, cycloaddition reactions, rearrangementreactions and any other known organic reactions that involve afunctional group.

A polymer, according to the present invention, may have one or morehydrophobic moiety residues, whereby at least one is linked to one aminoacid at one end and to another amino acid residue at another end, andanother may elongate the polymeric chain by being linked to either oneof the termini, i.e., the N-alpha of a terminal amino acid residueand/or the C-alpha of a terminal amino acid residue. Optionally, asecond hydrophobic moiety may be linked to the side-chain of an aminoacid residue in the polymer.

The polymer, according to the present invention, preferably includesfrom 2 to 50 amino acid residues. More preferably, the polymer includesfrom 2 to 12 amino acid residues, more preferably from 4 to 8 amino acidresidues and more preferably from 5 to 7 amino acid residues.

The net positive charge of the polymer is maintained by having one ormore positively charged amino acid residues in the polymer, optionallyin addition to the positively charged N-terminus amine, when present inits free form.

In one preferred embodiment of the present invention, all the amino acidresidues in the polymer are positively charged amino acid residues. Anexemplary polymer according to this embodiment includes a plurality oflysine residues.

As used herein the phrase “positively charged amino acid” describes ahydrophilic amino acid with a side chain pKa value of greater than 7,namely a basic amino acid. Basic amino acids typically have positivelycharged side chains at physiological pH due to association with ahydronium ion. Naturally occurring (genetically encoded) basic aminoacids include lysine (Lys, K), arginine (Arg, R) and histidine (His, H),while non-natural (non-genetically encoded, or non-standard) basic aminoacids include, for example, ornithine, 2,3,-diaminopropionic acid,2,4-diaminobutyric acid, 2,5,6-triaminohexanoic acid,2-amino-4-guanidinobutanoic acid, and homoarginine.

In one embodiment of the present invention, each of the components inthe polymer according to the present embodiments is preferably linked tothe other by a peptide bond.

The term “peptide bond” as used herein refers to an amide group, namely,a —(C═O)NH— group, which is typically formed by a condensation reactionbetween a carboxylic group and an amine group, as these terms aredefined herein.

However, the polymers of the present embodiments may have other bondslinking the various components in the polymeric structure. Suchnon-peptidic bonds may render the polymer more stable while in a body ormore capable of penetrating into cells. Thus, peptide bonds (—(C═O)NH—)within the polymer may be replaced, for example, by N-methylated amidebonds (—(C═O)NCH₃—), ester bonds (—C(R)H—C(═O)—O—C(R)—N—), ketomethylenbonds (—C(═O)CH₂—), aza bonds (—NH—N(R)—C(═O)—), wherein R is any alkyl,e.g., methyl, carba bonds (—CH₂—NH—), hydroxyethylene bonds(—CH(OH)—CH₂—), thioamide bonds (—CS—NH—), olefinic double bonds(—CH═CH—), retro amide bonds (—NH—(C═O)—), peptide derivatives(—N(R)—CH₂—C(═O)—), wherein R is the “normal” side chain, naturallypresented on the carbon atom. These modifications can occur at any ofthe bonds along the polymer chain and even several (2-3) at the sametime.

In a preferred embodiment, all of the bonds in the polymer, linking theamino acid residues and hydrophobic moiety residues to each other, arepeptide bonds. For example, in one embodiment, the polymer is made of anamino acid residue linked by a peptide bond to a hydrophobic moietyresidue which in turn is linked to a second amino acid residue byanother peptide bond. In another example, the polymer of the previousexample is elongated by a second hydrophobic moiety residue which islinked to any one of the N- or C-termini by a peptide bond, etcetera.

The polymer, according to the present invention, preferably comprisesfrom 1 to 50 hydrophobic moiety residues. More preferably, the polymercomprises from 1 to 12 hydrophobic moiety residues, more preferably from4 to 10 hydrophobic moiety residues and more preferably from 6 to 8hydrophobic moiety residues.

The hydrophobic moieties that are used in the context of this and otheraspects of the present invention preferably have one or more hydrocarbonchains, and are capable of linking to one or two other components in thepolymer (e.g., one or two of an amino acid residue and anotherhydrophobic moiety) via two peptide bonds. These moieties thereforepreferably have a carboxylic group at one end of the hydrocarbon chain(for linking a free amine group) and an amine group at the other (forlinking a carboxylic acid group).

The hydrocarbon chain connecting the carboxylic and amine groups in sucha hydrophobic moiety preferably has from 4 to 30 carbon atoms.

In a preferred embodiment of the present invention, the hydrophobicmoiety residue is a fatty acid residue wherein the hydrocarbon chain canbe unbranched and saturated, branched and saturated, unbranched andunsaturated or branched and unsaturated. More preferably the hydrocarbonchain of the fatty acid residue is an unbranched and saturated chainhaving from 4 to 30 carbon atoms, preferably from 4 to 20 carbon atoms.Non-limiting example of such fatty acid residues are butyric acidresidue, caprylic acid residue and lauric acid residue.

In a more preferred embodiment, the fatty acid residue has an amine onthe distal carbon of the hydrocarbon chain (with respect to thecarboxylic acid group). Such a fatty acid residue is referred to hereinas an ω-amino fatty acid residue. Again here the hydrocarbon chain ofthe ω-amino fatty acid residue may have from 4 to 30 carbon atoms.

Non-limiting example of such ω-amino fatty acids are 4-amino-butyricacid, 6-amino-caproic acid, 8-amino-caprylic acid, 10-amino-capric acid,12-amino-lauric acid, 14-amino-myristic acid, 16-amino-palmitic acid,18-amino-stearic acid, 18-amino-oleic acid, 16-amino-palmitoleic acid,18-amino-linoleic acid, 18-amino-linolenic acid and 20-amino-arachidonicacid.

According to a preferred embodiment of the present invention, thehydrophobic moiety is selected from the group consisting of4-amino-butyric acid, 8-amino-caprylic acid and 12-amino-lauric acid andmore preferably is 8-amino-caprylic acid and 12-amino-lauric acid.

The polymers described herein can be collectively represented by thefollowing general formula I:

X-W₀-[A₁-Z₁-D₁]-W₁-[A₂-Z₂-D₂]-W₂- . . . [An-Zn-Dn]-Wn-Y  Formula I

wherein:

n is an integer from 2 to 50, preferably from 2 to 12 and morepreferably from 2 to 8;

A₁, A₂, . . . , An are each independently an amino acid residue,preferably a positively charged amino acid residue, more preferably allof A₁, A₂, . . . , An are positively charged amino acid residues asdiscussed hereinabove, such as histidine residues, lysine residues,ornithine residues and arginine residues, and most preferably all thepositively charged amino acid residues are lysine residues;

D₁, D₂, . . . , Dn are each independently a hydrophobic moiety residue,as defined and discussed hereinabove, or absent, provided that at leastone such hydrophobic moiety residue exists in the polymer, preferably atleast one of the hydrophobic moiety residues is a ω-amino-fatty acidresidue;

Connecting each monomer of the residue are linking moieties, denoted Z₁,Z₂, . . . , Zn and W₀, W₁, W₂, . . . , Wn, each of which independentlylinking an amino acid residue and a hydrophobic moiety residue orabsent, preferably at least one of the linking moieties is a peptidebond and most preferable all the linking moieties are peptide bonds;

The fringes of the polymer, denoted X and Y, may each independently behydrogen, an amine, an amino acid residue, a hydrophobic moiety residue,is another polymer having the general Formula I or absent.

As discussed above, one or more of the hydrophobic moiety residues maybe attached to a side chain of one or more of the amino acid residues ofthe polymer, i.e., act as a branch of the main polymer.

The presently most preferred polymers are polymers in which n is aninteger from 5 to 7, the amino acid residues are all lysine residues,the hydrophobic moiety residues are all 8-amino-caprylic acid residues,X is a hydrophobic moiety such as, for example, a fatty acid residue (adodecanoic acid residue), and/or Y is amine or absent.

Particularly preferred polymers according to the present embodiments arethose having the Formulae hereinbelow:

which is also referred to herein as C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45); and

which is also referred to herein as C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43).

The polymers according to the present embodiments can be readilysynthesized. For example, polymers in which the linking moieties arepeptide bonds, and hence resemble natural and synthetic peptides in thisrespect, can be prepared by classical methods known in the art forpeptide syntheses. Such methods include, for example, standard solidphase techniques. The standard methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensation,classical solution synthesis, and even by recombinant DNA technology.See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149 (1963), incorporatedherein by reference. Solid phase peptide synthesis procedures are wellknown in the art and further described by John Morrow Stewart and JanisDillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce ChemicalCompany, 1984).

The polymers of the present invention can be purified, for example, bypreparative high performance liquid chromatography [Creighton T. (1983)Proteins, structures and molecular principles. WH Freeman and Co. N.Y.].

Apart from having beneficial antimicrobial activity per se, as detailedherein, the polymers of the present invention may include an additionalactive agent such as a labeling agent and/or a therapeutically activeagent attached thereto. The conjugation of the active agent to a polymerof the present invention can provide a dual utility for the polymer.When the additional active agent is a labeling agent, the conjugationthereof to an antimicrobial polymer of the present invention, having ahigh affinity to microbial cells, can assist in the location, diagnosisand targeting of microbial growth loci in a host. When the additionalactive agent is a therapeutically active agent, the conjugation thereofto an antimicrobial polymer of the present invention will exert a dualand possibly synergistic antimicrobial activity.

According to preferred embodiments of the present invention, the one ormore active agents may be attached to the polymer at any substitutableposition. Examples of such substitutable positions include, withoutlimitation, a side chain of any one or more of the amino acid residuesin the polymer, any one of the linking moieties of the polymer, any oneof the N- and C-termini of the polymer and any one or more of thehydrophobic moiety residues in the polymer.

Hence, as used herein, the phrase “a therapeutically active agent”describes a chemical substance, which exhibit a therapeutic activitywhen administered to a subject

As used herein, the phrase “labeling agent” refers to a detectablemoiety or a probe and includes, for example, chromophores, fluorescentcompounds, phosphorescent compounds, heavy metal clusters, andradioactive labeling compounds, as well as any other known detectablemoieties.

Labeling of microbial growth loci in a host is critical for thediagnosis and efficient targeting of the photogenic microorganism andtreatment thereof.

Adding a therapeutically active agent to the polymer can provide asolution for many deficiencies of presently known therapeutically activeagent against photogenic microorganisms, such as resistance of thephotogenic microorganism to the therapeutically active agent,specificity of the therapeutically active agent to photogenicmicroorganism and general efficacy weakness. The polymers of the presentinvention can exhibit not only antimicrobial activity per se by virtueof their structure and chemical properties, but can also providetargeting capacity as a delivery vehicle to a presently knowtherapeutically active agents and further provide membrane permeabilityto presently know therapeutically active agents due to their capabilityto exert disturbance in the membrane structure of photogenicmicroorganisms.

Non-limiting examples of therapeutically active agents that can bebeneficially used in this and other contexts of the present inventioninclude, without limitation, one or more of an agonist residue, an aminoacid residue, an analgesic residue, an antagonist residue, an antibioticagent residue, an antibody residue, an antidepressant agent, an antigenresidue, an anti-histamine residue, an anti-hypertensive agent, ananti-inflammatory drug residue, an anti-metabolic agent residue, anantimicrobial agent residue, an antioxidant residue, ananti-proliferative drug residue, an antisense residue, achemotherapeutic drug residue, a co-factor residue, a cytokine residue,a drug residue, an enzyme residue, a growth factor residue, a heparinresidue, a hormone residue, an immunoglobulin residue, an inhibitorresidue, a ligand residue, a nucleic acid residue, an oligonucleotideresidue, a peptide residue, a phospholipid residue, a prostaglandinresidue, a protein residue, a toxin residue, a vitamin residue and anycombination thereof.

The combined therapeutic effect is particularly advantageous when thetherapeutically active agent is an antimicrobial or an antibiotic agent.The combined activity of the polymers of the present invention and thatof an additional antimicrobial/antibiotic agent may provide theantimicrobial/antibiotic agent the capacity to overcome the knownlimitations of these drugs such as targeting, specificity, efficacy,drug-resistance etcetera. Synergism may also be achieved.

Non-limiting examples of antimicrobial and antibiotic agents that aresuitable for use in this context of the present invention include,without limitation, mandelic acid, 2,4-dichlorobenzenemethanol,4-[bis(ethylthio)methyl]-2-methoxyphenol, 4-epi-tetracycline,4-hexylresorcinol, 5,12-dihydro-5,7,12,14-tetrazapentacen,5-chlorocarvacrol, 8-hydroxyquinoline, acetarsol, acetylkitasamycin,acriflavin, alatrofloxacin, ambazon, amfomycin, amikacin, amikacinsulfate, aminoacridine, aminosalicylate calcium, aminosalicylate sodium,aminosalicylic acid, ammoniumsulfobituminat, amorolfin, amoxicillin,amoxicillin sodium, amoxicillin trihydrate, amoxicillin-potassiumclavulanate combination, amphotericin B, ampicillin, ampicillin sodium,ampicillin trihydrate, ampicillin-sulbactam, apalcillin, arbekacin,aspoxicillin, astromicin, astromicin sulfate, azanidazole,azidamfenicol, azidocillin, azithromycin, azlocillin, aztreonam,bacampicillin, bacitracin, bacitracin zinc, bekanamycin, benzalkonium,benzethonium chloride, benzoxonium chloride, berberine hydrochloride,biapenem, bibrocathol, biclotymol, bifonazole, bismuth subsalicylate,bleomycin antibiotic complex, bleomycin hydrochloride, bleomycinsulfate, brodimoprim, bromochlorosalicylanilide, bronopol,broxyquinolin, butenafine, butenafine hydrochloride, butoconazol,calcium undecylenate, candicidin antibiotic complex, capreomycin,carbenicillin, carbenicillin disodium, carfecillin, carindacillin,carumonam, carzinophilin, caspofungin acetate, cefacetril, cefaclor,cefadroxil, cefalexin, cefalexin hydrochloride, cefalexin sodium,cefaloglycin, cefaloridine, cefalotin, cefalotin sodium, cefamandole,cefamandole nafate, cefamandole sodium, cefapirin, cefapirin sodium,cefatrizine, cefatrizine propylene glycol, cefazedone, cefazedone sodiumsalt, cefazolin, cefazolin sodium, cefbuperazone, cefbuperazone sodium,cefcapene, cefcapene pivoxil hydrochloride, cefdinir, cefditoren,cefditoren pivoxil, cefepime, cefepime hydrochloride, cefetamet,cefetamet pivoxil, cefixime, cefmenoxime, cefmetazole, cefmetazolesodium, cefminox, cefminox sodium, cefmolexin, cefodizime, cefodizimesodium, cefonicid, cefonicid sodium, cefoperazone, cefoperazone sodium,ceforanide, cefoselis sulfate, cefotaxime, cefotaxime sodium, cefotetan,cefotetan disodium, cefotiam, cefotiam hexetil hydrochloride, cefotiamhydrochloride, cefoxitin, cefoxitin sodium, cefozopran hydrochloride,cefpiramide, cefpiramide sodium, cefpirome, cefpirome sulfate,cefpodoxime, cefpodoxime proxetil, cefprozil, cefquinome, cefradine,cefroxadine, cefsulodin, ceftazidime, cefteram, cefteram pivoxil,ceftezole, ceftibuten, ceftizoxime, ceftizoxime sodium, ceftriaxone,ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime sodium,cetalkonium chloride, cetrimide, cetrimonium, cetylpyridinium,chloramine T, chloramphenicol, chloramphenicol palmitate,chloramphenicol succinate sodium, chlorhexidine, chlormidazole,chlormidazole hydrochloride, chloroxylenol, chlorphenesin,chlorquinaldol, chlortetracycline, chlortetracycline hydrochloride,ciclacillin, ciclopirox, cinoxacin, ciprofloxacin, ciprofloxacinhydrochloride, citric acid, clarithromycin, clavulanate potassium,clavulanate sodium, clavulanic acid, clindamycin, clindamycinhydrochloride, clindamycin palmitate hydrochloride, clindamycinphosphate, clioquinol, cloconazole, cloconazole monohydrochloride,clofazimine, clofoctol, clometocillin, clomocycline, clotrimazol,cloxacillin, cloxacillin sodium, colistin, colistin sodiummethanesulfonate, colistin sulfate, cycloserine, dactinomycin,danofloxacin, dapsone, daptomycin, daunorubicin, DDT, demeclocycline,demeclocycline hydrochloride, dequalinium, dibekacin, dibekacin sulfate,dibrompropamidine, dichlorophene, dicloxacillin, dicloxacillin sodium,didecyldimethylammonium chloride, dihydrostreptomycin,dihydrostreptomycin sulfate, diiodohydroxyquinolin, dimetridazole,dipyrithione, dirithromycin, DL-menthol, D-menthol,dodecyltriphenylphosphonium bromide, doxorubicin, doxorubicinhydrochloride, doxycycline, doxycycline hydrochloride, econazole,econazole nitrate, enilconazole, enoxacin, enrofloxacin, eosine,epicillin, ertapenem sodium, erythromycin, erythromycin estolate,erythromycin ethyl succinate, erythromycin lactobionate, erythromycinstearate, ethacridine, ethacridine lactate, ethambutol, ethanoic acid,ethionamide, ethyl alcohol, eugenol, exalamide, faropenem,fenticonazole, fenticonazole nitrate, fezatione, fleroxacin, flomoxef,flomoxef sodium, florfenicol, flucloxacillin, flucloxacillin magnesium,flucloxacillin sodium, fluconazole, flucytosine, flumequine,flurithromycin, flutrimazole, fosfomycin, fosfomycin calcium, fosfomycinsodium, framycetin, framycetin sulphate, furagin, furazolidone,fusafungin, fusidic acid, fusidic acid sodium salt, gatifloxacin,gemifloxacin, gentamicin antibiotic complex, gentamicin c la, gentamycinsulfate, glutaraldehyde, gramicidin, grepafloxacin, griseofulvin,halazon, haloprogine, hetacillin, hetacillin potassium, hexachlorophene,hexamidine, hexetidine, hydrargaphene, hydroquinone, hygromycin,imipenem, isepamicin, isepamicin sulfate, isoconazole, isoconazolenitrate, isoniazid, isopropanol, itraconazole, josamycin, josamycinpropionate, kanamycin, kanamycin sulphate, ketoconazole, kitasamycin,lactic acid, lanoconazole, lenampicillin, leucomycin A1, leucomycin A13,leucomycin A4, leucomycin A5, leucomycin A6, leucomycin A7, leucomycinA8, leucomycin A9, levofloxacin, lincomycin, lincomycin hydrochloride,linezolid, liranaftate, 1-menthol, lomefloxacin, lomefloxacinhydrochloride, loracarbef, lymecyclin, lysozyme, mafenide acetate,magnesium monoperoxophthalate hexahydrate, mecetronium ethylsulfate,mecillinam, meclocycline, meclocycline sulfosalicylate, mepartricin,merbromin, meropenem, metalkonium chloride, metampicillin, methacycline,methenamin, methyl salicylate, methylbenzethonium chloride,methylrosanilinium chloride, meticillin, meticillin sodium,metronidazole, metronidazole benzoate, mezlocillin, mezlocillin sodium,miconazole, miconazole nitrate, micronomicin, micronomicin sulfate,midecamycin, minocycline, minocycline hydrochloride, miocamycin,miristalkonium chloride, mitomycin c, monensin, monensin sodium,morinamide, moxalactam, moxalactam disodium, moxifloxacin, mupirocin,mupirocin calcium, nadifloxacin, nafcillin, nafcillin sodium, naftifine,nalidixic acid, natamycin, neomycin a, neomycin antibiotic complex,neomycin C, neomycin sulfate, neticonazole, netilmicin, netilmicinsulfate, nifuratel, nifuroxazide, nifurtoinol, nifurzide, nimorazole,niridazole, nitrofurantoin, nitrofurazone, nitroxolin, norfloxacin,novobiocin, nystatin antibiotic complex, octenidine, ofloxacin,oleandomycin, omoconazol, orbifloxacin, ornidazole, ortho-phenylphenol,oxacillin, oxacillin sodium, oxiconazole, oxiconazole nitrate, oxoferin,oxolinic acid, oxychlorosene, oxytetracycline, oxytetracycline calcium,oxytetracycline hydrochloride, panipenem, paromomycin, paromomycinsulfate, pazufloxacine, pefloxacin, pefloxacin mesylate, penamecillin,penicillin G, penicillin G potassium, penicillin G sodium, penicillin V,penicillin V calcium, penicillin V potassium, pentamidine, pentamidinediisetionate, pentamidine mesilas, pentamycin, phenethicillin, phenol,phenoxyethanol, phenylmercuriborat, PHMB, phthalylsulfathiazole,picloxydin, pipemidic acid, piperacillin, piperacillin sodium,pipercillin sodium-tazobactam sodium, piromidic acid, pivampicillin,pivcefalexin, pivmecillinam, pivmecillinam hydrochloride, policresulen,polymyxin antibiotic complex, polymyxin B, polymyxin B sulfate,polymyxin B1, polynoxylin, povidone-iodine, propamidin, propenidazole,propicillin, propicillin potassium, propionic acid, prothionamide,protiofate, pyrazinamide, pyrimethamine, pyrithion, pyrrolnitrin,quinoline, quinupristin-dalfopristin, resorcinol, ribostamycin,ribostamycin sulfate, rifabutin, rifampicin, rifamycin, rifapentine,rifaximin, ritiometan, rokitamycin, rolitetracycline, rosoxacin,roxithromycin, rufloxacin, salicylic acid, secnidazol, seleniumdisulphide, sertaconazole, sertaconazole nitrate, siccanin, sisomicin,sisomicin sulfate, sodium thiosulfate, sparfloxacin, spectinomycin,spectinomycin hydrochloride, spiramycin antibiotic complex, spiramycinb, streptomycin, streptomycin sulphate, succinylsulfathiazole,sulbactam, sulbactam sodium, sulbenicillin disodium, sulbentin,sulconazole, sulconazole nitrate, sulfabenzamide, sulfacarbamide,sulfacetamide, sulfacetamide sodium, sulfachlorpyridazine, sulfadiazine,sulfadiazine silver, sulfadiazine sodium, sulfadicramide,sulfadimethoxine, sulfadoxine, sulfaguanidine, sulfalene, sulfamazone,sulfamerazine, sulfamethazine, sulfamethazine sodium, sulfamethizole,sulfamethoxazole, sulfamethoxazol-trimethoprim, sulfamethoxypyridazine,sulfamonomethoxine, sulfamoxol, sulfanilamide, sulfaperine,sulfaphenazol, sulfapyridine, sulfaquinoxaline, sulfasuccinamide,sulfathiazole, sulfathiourea, sulfatolamide, sulfatriazin,sulfisomidine, sulfisoxazole, sulfisoxazole acetyl, sulfonamides,sultamicillin, sultamicillin tosilate, tacrolimus, talampicillinhydrochloride, teicoplanin A2 complex, teicoplanin A2-1, teicoplaninA2-2, teicoplanin A2-3, teicoplanin A2-4, teicoplanin A2-5, teicoplaninA3, teicoplanin antibiotic complex, telithromycin, temafloxacin,temocillin, tenoic acid, terbinafine, terconazole, terizidone,tetracycline, tetracycline hydrochloride, tetracycline metaphosphate,tetramethylthiuram monosulfide, tetroxoprim, thiabendazole,thiamphenicol, thiaphenicol glycinate hydrochloride, thiomersal, thiram,thymol, tibezonium iodide, ticarcillin, ticarcillin-clavulanic acidmixture, ticarcillin disodium, ticarcillin monosodium, tilbroquinol,tilmicosin, tinidazole, tioconazole, tobramycin, tobramycin sulfate,tolciclate, tolindate, tolnaftate, toloconium metilsulfat, toltrazuril,tosufloxacin, triclocarban, triclosan, trimethoprim, trimethoprimsulfate, triphenylstibinsulfide, troleandomycin, trovafloxacin, tylosin,tyrothricin, undecoylium chloride, undecylenic acid, vancomycin,vancomycin hydrochloride, viomycin, virginiamycin antibiotic complex,voriconazol, xantocillin, xibornol and zinc undecylenate.

Major parts of the polymers of the present embodiments are based on arepetitive element consisting of a conjugate between an amino acid and abi-functional hydrophobic moiety. The conjugate may repeat several timesin the sequence of the polymer and/or be interrupted and/or flanked by adifference types of conjugates or by single or repeats of amino acidresidues and single or repeats of hydrophobic moiety residues.

Hence, according to another aspect of the present invention, there isprovided a conjugate which includes an amino acid residue and ahydrophobic moiety residue, as defined and described hereinabove,attached to the N-alpha or the C-alpha of the amino acid residue. Thehydrophobic moiety residue in the conjugate of the present invention isdesigned such that is it capable of forming a bond with an N-alpha or aC-alpha of an additional amino acid residue. Preferably, the hydrophobicmoiety residue is conjugated to the amino acid residue via a peptidebond.

The hydrophobic moiety of the conjugate of the present invention ishaving a bi-functional design which allows the conjugate to serve as apolymerizable conjugate that can form a part of the polymers describedand presented herein. Preferably, the hydrophobic moiety which forms apart of the conjugate is having a bi-functionality in the form of acarboxylic group at one end thereof and an amine group at the other endthereof.

Hence, according to another aspect of the present invention, there isprovided a process of preparing the conjugate described hereinabove, thegeneral process is based on providing an amino acid, preferably theamino acid is a positively charged amino acid, such as histidine,lysine, ornithine and arginine; providing a hydrophobic moiety asdefined and discussed hereinabove having a first functional group thatis capable of reacting with an N-alpha of an amino acid residue and asecond functional group capable of reacting with a C-alpha of an aminoacid; linking the first functional group in the hydrophobic moiety tothe amino acid via the N-alpha of the amino acid; or linking the secondfunctional group in the hydrophobic moiety to the amino acid via theC-alpha of the amino acid.

Preferably, the link between the N-alpha or the C-alpha of the aminoacid and the hydrophobic moiety is via a peptide bond.

In order to form a peptide bond linking the amino acid to thehydrophobic moiety, the hydrophobic moiety preferably has a carboxylicgroup at one end thereof and an amine group at the other end thereof.

The antimicrobial polymers as described herein can be beneficiallyutilized in the treatment of pathogenic microorganism infections, asthese are defined hereinbelow. As demonstrated in the Example sectionthat follows, such polymers are by themselves capable of exertingantimicrobial activity. The option to include an additionaltherapeutically active agent may thus act synergistically as toxicagents against various bacteria, fungi and other microorganisms.

Herein throughout, the phrase “pathogenic microorganism” is used todescribe any microorganism which can cause a disease or disorder in ahigher organism, such as mammals in general and a human in particular.The pathogenic microorganism may belong to any family of organisms suchas, but not limited to prokaryotic organisms, eubacterium,archaebacterium, eukaryotic organisms, yeast, fungi, algae, protozoan,and other parasites. Non-limiting examples of pathogenic microorganismare Plasmodium falciparum and related malaria-causing protozoanparasites, Acanthamoeba and other free-living amoebae, Aeromonashydrophila, Anisakis and related worms, Acinetobacter baumanii, Ascarislumbricoides, Bacillus cereus, Brevundimonas diminuta, Campylobacterjejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidiumparvum, Cyclospora cayetanensis, Diphyllobothrium, Entamoebahistolytica, certain strains of Escherichia coli, Eustrongylides,Giardia lamblia, Klebsiella pneumoniae, Listeria monocytogenes,Nanophyetus, Plesiomonas shigelloides, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella, Serratia odorifera, Shigella, Staphylococcusaureus, Stenotrophomonas maltophilia, Streptococcus, Trichuristrichiura, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificusand other vibrios, Yersinia enterocolitica, Yersinia pseudotuberculosisand Yersinia kristensenii.

Hence, according to another aspect of the present invention, there isprovided a method of treating a medical condition associated with apathogenic microorganism, the method includes administering to a subjectin need thereof a therapeutically effective amount of one or more of thepolymers, as described hereinabove

As used herein, the terms “treating” and “treatment” includesabrogating, substantially inhibiting, slowing or reversing theprogression of a condition, substantially ameliorating clinical oraesthetical symptoms of a condition or substantially preventing theappearance of clinical or aesthetical symptoms of a condition.

As used herein, the phrase “therapeutically effective amount” describesan amount of the composite being administered which will relieve to someextent one or more of the symptoms of the condition being treated.

The method of treatment, according to an embodiment of the presentinvention, may include the administration of an additionaltherapeutically active agent, as this is defined and discussedhereinabove.

As mentioned above and demonstrated in the Example section that follows,the antimicrobial polymers of the present invention, alone or incombination with any other therapeutically active agents, can bedesigned and utilized to destroy pathological microorganisms. Thedestruction of a pathogenic microorganism is effected by selectivelydestructing a portion of the cells of a pathogenic microorganism. Whilemost known antibiotics act by interfering selectively with thebiosynthesis of one or more of the molecular constituents of thecell-membrane, proteins or nucleic acids, the polymers of the presentinvention also act by binding and disrupting the outer membrane of thepathogenic microorganism cells. Disrupting the outer membrane of a cellcauses its death due to membrane depolarization, leakage of metabolitesand/or total loss of cell integrity; therefore the polymers of thepresent invention also act directly as effective antimicrobial agents bydisrupting the metabolism and/or the multiplication processes of thepathogenic microorganism.

As mentioned above and demonstrated in the Example section that follows,the polymers presented herein may act as antimicrobial agents which donot evoke the appearance of resistance thereto. The possible developmentof resistance to the polymers of the present invention was tested bymeasuring the minimal inhibitory concentration (MIC) levels followingmultiple exposures of the bacteria to exemplary polymers according tothe present invention. The results obtained in theantimicrobial-resistance studies in bacteria presented hereinbelow,showed that exposing bacteria, and even strains that already developedresistance to classical antibiotics, to the antimicrobial polymerspresented herein did not result in development of resistance.

As is further mentioned above and demonstrated in the Example sectionthat follows, the polymers presented herein are non-toxic to mammals.

As is further demonstrated in the Examples section that follows, thepolymers of the present invention can act synergistically with anotherantibiotic or other therapeutically active agent by permeabilizing thecells of the pathogenic microorganism; hence exhibit additionally anindirect antimicrobial activity. The results presented hereinbelowpermit the conclusion that the polymers of the present invention arepotent outer-membrane disintegrating agents. The permeabilizing actionof the polymers can increase the uptake of other therapeutically activeagents and therefore should be able to potentiate the apparentantimicrobial activity of other drugs and antibiotics.

Medical conditions associated with a pathogenic microorganism includeinfections, infestation, contaminations and transmissions by or ofpathogenic microorganism. In general, a disease causing infection is theinvasion into the tissues of a plant or an animal by pathogenicmicroorganisms. The invasion of body tissues by parasitic worms andother higher pathogenic organisms is commonly referred to asinfestation.

Invading organisms such as bacteria produce toxins that damage hosttissues and interfere with normal metabolism; some toxins are actuallyenzymes that break down host tissues. Other bacterial substances mayinflict their damage by destroying the host's phagocytes, rendering thebody more susceptible to infections by other pathogenic microorganisms.Substances produced by many invading organisms cause allergicsensitivity in the host. Infections may be spread via respiratorydroplets, direct contact, contaminated food, or vectors, such asinsects. They can also be transmitted sexually and from mother to fetus.

Diseases caused by bacterial infections typically include, for example,actinomycosis, anthrax, aspergillosis, bacteremia, bacterial skindiseases, bartonella infections, botulism, brucellosis, burkholderiainfections, campylobacter infections, candidiasis, cat-scratch disease,chlamydia infections, cholera, clostridium infections,coccidioidomycosis, cryptococcosis, dermatomycoses, diphtheria,ehrlichiosis, epidemic louse borne typhus, Escherichia coli infections,fusobacterium infections, gangrene, general infections, general mycoses,gonorrhea, gram-negative bacterial infections, gram-positive bacterialinfections, histoplasmosis, impetigo, klebsiella infections,legionellosis, leprosy, leptospirosis, listeria infections, lymedisease, malaria, maduromycosis, melioidosis, mycobacterium infections,mycoplasma infections, necrotizing fasciitis, nocardia infections,onychomycosis, ornithosis, pneumococcal infections, pneumonia,pseudomonas infections, Q fever, rat-bite fever, relapsing fever,rheumatic fever, rickettsia infections, Rocky-mountain spotted fever,salmonella infections, scarlet fever, scrub typhus, sepsis, sexuallytransmitted bacterial diseases, staphylococcal infections, streptococcalinfections, surgical site infection, tetanus, tick-borne diseases,tuberculosis, tularemia, typhoid fever, urinary tract infection, vibrioinfections, yaws, yersinia infections, Yersinia pestis plague, zoonosesand zygomycosis.

The polymers of the present embodiments can therefore be used to treatmedical conditions caused by pathogenic microorganisms by virtue oftheir anti-microbial effects inflicted upon the pathogenicmicroorganisms by one of the abovementioned mechanism which mostly stemfrom their specific and selective affinity to the membrane of thepathogenic microorganism, and relative undamaging effect they have onmammalian cell, as demonstrated for red blood cells and presented in theExamples section that follows. This affinity can be used to weaken,disrupt, puncture, melt, fuse and/or mark the membrane of a pathogenicmicroorganism.

The pathogenic microorganism may be destroyed directly by the disruptionof its membrane as demonstrated and presented for a series of bacterialstrains in the Examples section that follows, or be weakened so as toallow the innate immune system to destroy it or slow down its metabolismand therefore its reproduction so as to allow the innate immune systemto overcome the infection.

The pathogenic microorganism may be destroyed by the disruption of itsmembrane so as to allow a therapeutically active agent, such as anantibiotic agent, to more easily penetrate the cell of the microorganismand afflict its activity thereon.

The latter capacity of the antimicrobial polymer of the presentinvention to assist the penetration of another therapeutically activeagent into the cells of the pathogenic microorganism can be utilized totreat many infectious diseases, such as, for example, malaria.

The experimental results presented in the Examples section that followssuggests that secondary structure might not be an absolute prerequisitefor antimicrobial properties. On another hand, evident from theseresults stems that the only property which is shared by all typicalAMPs, and also shared by the polymers of the present invention, is therelative abundance of both hydrophobic and positively charged amino acidresidues. Thus, according to the present invention, the antimicrobialpolymers presented are endowed with varied positive charge andhydrophobicity and substantially lack secondary structure.

Malaria, also called jungle fever, paludism and swamp fever, is aninfectious disease characterized by cycles of chills, fever, andsweating, caused by the parasitic infection of red blood cells by theprotozoan parasite, Plasmodium (one of the Apicomplexa), which istransmitted by the bite of an infected vector for human malarialparasite, a female Anopheles mosquito. Of the four types of malaria, themost life-threatening type is falciparum malaria. The other three typesof malaria, vivax, malariae, and ovale, are generally less serious andare not life-threatening. Malaria, the deadliest infectious disease yetto be beaten, causes about half a billion infections and between one andtwo millions deaths annually, mainly in the tropics and sub-SaharanAfrica. The Plasmodium falciparum variety of the parasite accounts for80% of cases and 90% of deaths. The stickiness of the red blood cells isparticularly pronounced in P. falciparum malaria and this is the mainfactor giving rise to hemorrhagic complications of malaria.

To date there is no absolute cure for malaria. If diagnosed early,malaria can be alleviated, but prevention still more effective thantreatment, thus substances that inhibit the parasite are widely used byvisitors to the tropics. Since the 17^(th) century quinine has been theprophylactic of choice for malaria. The development of quinacrine,chloroquine, and primaquine in the 20^(th) century reduced the relianceon quinine. These anti-malarial medications can be taken preventively,which is recommended for travelers to affected regions.

Unfortunately as early as the 1960s several strains of the malarialparasite developed resistance to chloroquine. This development ofresistance, plus the growing immunity of mosquitoes to insecticides, hascaused malaria to become one the of world's leading re-emerginginfectious diseases. Mefloquine may be used in areas where the diseasehas become highly resistant to chloroquine, but some strains are nowresistant to it and other drugs. Artemisinin (derived from sweetwormwood) in combination with other drugs is now in many cases thepreferred treat for resistant strains. Malarone (atovaquone andproguanil) is also used for resistant strains. Vaccines against malariaare still experimental.

While reducing the present invention to practice, the present inventorshave prepared and successfully used these anti-microbial polymers asanti-malarial agents with reduced hemolysis effect as demonstrated inthe Examples section that follows. It is shown that the polymers of thepresent invention were able to kill the parasite in a manner that isclearly dissociated from lysis of the host cell. These polymers wereable to enter the infected cell but to selectively permeabilize theparasite cell membrane. These results are best explained by thedifferential interaction of the peptides-like polymer with the distinctproperties of the structure and composition of the membranes ofintra-erythrocytic malaria parasite Plasmodium falciparum as compared tothose of the host and normal red blood cells. These findings alsoestablished that the membrane active polymers of the present inventioncould be engineered to act specifically on the membrane of theintracellular parasite to perturb its functions. The polymers of thepresent invention can therefore overcome the problem of parasiticresistance to various anti-malarial agents by, for example, weakeningthe parasite's membrane and enabling the anti-malarial agents topenetrate the parasite's membrane more rapidly.

Therefore, a preferred embodiment of the present invention is the use ofthe antimicrobial polymers as an anti-malarial agent, either per-se orin combination with a presently used anti-malarial agent or any otheranti-parasitic agent, as exemplified in the Examples section thatfollows.

In any of the aspects of the present invention, the antimicrobialpolymers of the present invention can be utilized either per se, or asan active ingredient that forms a part of a pharmaceutical composition,with or without an additional therapeutically active agent, and apharmaceutically acceptable carrier.

Hence, according to still another aspect of the present invention, thereare provided pharmaceutical compositions, which comprise one or more ofthe polymers of the present invention as described above having anantimicrobial activity and a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofthe antimicrobial polymer described herein, with other chemicalcomponents such as pharmaceutically acceptable and suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the silver-coated enzymesinto preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.Toxicity and therapeutic efficacy of the silver-coated enzymes describedherein can be determined by standard pharmaceutical procedures inexperimental animals, e.g., by determining the EC₅₀, the IC₅₀ and theLD₅₀ (lethal dose causing death in 50% of the tested animals) for asubject silver-coated enzyme. The data obtained from these activityassays and animal studies can be used in formulating a range of dosagefor use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising asilver-coated enzyme of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition ordiagnosis, as is detailed hereinabove.

Thus, according to an embodiment of the present invention, depending onthe selected polymers and the presence of additional active ingredients,the pharmaceutical compositions of the present invention are packaged ina packaging material and identified in print, in or on the packagingmaterial, for use in the treatment of a medical condition associatedwith a pathogenic microorganism, as is defined hereinabove and aparasite.

The pharmaceutical composition comprising a polymer of the presentinvention may further comprise at least one additional therapeuticallyactive agent, as this is defined and presented hereinabove.

The polymers of the present invention can be further beneficiallyutilized as active substances in various medical devices.

Hence, according to an additional aspect of the present invention thereis provided a medical device which includes one or more of the polymersof the present invention, described hereinabove, and a delivery systemconfigured for delivering the polymer(s) to a bodily site of a subject.

The medical devices according to the present invention are thereforeused for delivering to or applying on a desired bodily site the polymersof the present invention. The polymers can be incorporated in themedical devices either per se or as a part of a pharmaceuticalcomposition, as described hereinabove.

As used herein, the phrase “bodily site” includes any organ, tissue,membrane, cavity, blood vessel, tract, biological surface or muscle,which delivering thereto or applying thereon the polymers of the presentinvention is beneficial.

Exemplary bodily sites include, but are not limited to, the skin, adermal layer, the scalp, an eye, an ear, a mouth, a throat, a stomach, asmall intestines tissue, a large intestines tissue, a kidney, apancreas, a liver, the digestive system, the respiratory tract, a bonemarrow tissue, a mucosal membrane, a nasal membrane, the blood system, ablood vessel, a muscle, a pulmonary cavity, an artery, a vein, acapillary, a heart, a heart cavity, a male or female reproductive organand any visceral organ or cavity.

The medical devices according to this aspect of the present inventioncan be any medical device known in the art, including those defined andclassified, for example, by the Food and Drug Administration (FDA),e.g., Class I, II and III, depending e.g., on the condition and bodilysite being treated.

Thus, for example, in one embodiment of this aspect of the presentinvention, the medical device comprises a delivery system that isconfigured to deliver the polymer(s) by inhalation. Such inhalationdevices are useful for delivering the polymers of the present inventionto, e.g., the respiratory tract.

The delivery system in such medical devices may be based on any ofvarious suitable types of respiratory delivery systems which aresuitable for administering a therapeutically effective dose of thepolymer(s) of the present invention to a subject.

The inhalation device may be configured to deliver to the respiratorytract of the subject, preferably via the oral and/or nasal route, thecompound in the form of an aerosol/spray, a vapor and/or a dry powdermist. Numerous respiratory systems and methods of incorporatingtherapeutic agents therein, such as the polymers of the presentinvention, suitable for assembly of a suitable inhalation device arewidely employed by the ordinarily skilled artisan and are extensivelydescribed in the literature of the art (see, for example U.S. Pat. Nos.6,566,324, 6,571,790, 6,637,430, and 6,652,323; and the U.S. Food & DrugAdministration (USFDA) Center For Drug Evaluation and Research (CDER).

The respiratory delivery system may thus be, for example, an atomizer oraerosol generator such as a nebulizer inhaler, a dry powder inhaler(DPI) and a metered dose inhaler (MDI), an evaporator such as anelectric warmer and a vaporizer, and a respirator such as a breathingmachine, a body respirator (e.g., cuirass), a lung ventilator and aresuscitator.

In still another embodiment of this aspect of the present invention, themedical device is such that delivering the polymer(s) is effectedtransdermally. In this embodiment, the medical device is applied on theskin of a subject, so as to transdermally deliver the polymer(s) to theblood system.

Exemplary medical devices for transdermally delivering a polymeraccording to the present invention include, without limitation, anadhesive plaster and a skin patch. Medical devices for transdermal ortranscutaneous delivery of the polymer(s) typically further include oneor more penetration enhancers, for facilitating their penetrationthrough the epidermis and into the system.

According to another embodiment of this aspect of the present invention,the medical device is such that delivering the polymer(s) is effected bytopically applying the medical device on a biological surface of asubject. The biological surface can be, for example, a skin, scalp, aneye, an ear and a nail. Such medical devices can be used in thetreatment of various skin conditions and injuries, eye and earinfections and the like.

Exemplary medical devices for topical application include, withoutlimitation, an adhesive strip, a bandage, an adhesive plaster, a wounddressing and a skin patch.

In another embodiment of this aspect of the present invention, themedical device is such that delivering the polymer(s) is effected byimplanting the medical device in a bodily organ. As used herein, theterm “organ” further encompasses a bodily cavity.

The organ can be, for example, a pulmonary cavity, a heart or heartcavity, a bodily cavity, an organ cavity, a blood vessel, an artery, avein, a muscle, a bone, a kidney, a capillary, the space between dermallayers, an organ of the female or male reproductive system, an organ ofthe digestive tract and any other visceral organ.

The medical device according to this embodiment of the present inventiontypically includes a device structure in which a polymer according tothe present invention is incorporated. The polymer(s) can thus be, forexample, applied on, entrapped in or attached to (chemically,electrostatically or otherwise) the device structure.

The device structure can be, for example, metallic structure and thusmay be comprised of a biocompatible metal or mixture of metals (e.g.,gold, platinum).

Alternatively, the device structure may be comprised of otherbiocompatible matrices. These can include, for example, plastics,silicon, polymers, resins, and may include at least one component suchas, for example, polyurethane, cellulose ester, polyethylene glycol,polyvinyl acetate, dextran, gelatin, collagen, elastin, laminin,fibronectin, vitronectin, heparin, segmented polyurethane-urea/heparin,poly-L-lactic acid, fibrin, cellulose and amorphous or structured carbonsuch as in fullerenes, and any combination thereof.

In cases where a biodegradable implantable device is desired, the devicestructure can be comprised of a biocompatible matrix that isbiodegradable. Biodegradable matrices can include, for example,biodegradable polymers such as poly-L-lactic acid.

Optionally, the device structure may be comprised of biocompatiblemetal(s) coated with other biocompatible matrix.

Further optionally, in cases where a device which releases thepolymer(s) of the present invention in a controlled manner is desired,the device structure can be comprised of or coated with a biocompatiblematrix that functions as or comprises a slow release carrier. Thebiocompatible matrix can therefore be a slow release carrier which isdissolved, melted or liquefied upon implantation in the desired site ororgan.

Alternatively, the biocompatible matrix can be a pre-determined porousmaterial which entraps the polymer(s) in the pores. When implanted in adesired site, the polymer(s) diffuse out of the pores, whereby thediffusion rate is determined by the pores size and chemical nature.Further alternatively, the biocompatible matrix can comprise abiodegradable matrix, which upon degradation releases the polymer(s) ofthe present invention.

The polymer(s) of the present invention can be incorporated in thedevice structure by any methodology known in the art, depending on theselected nature of the device structure. For example, the polymer(s) canbe entrapped within a porous matrix, swelled or soaked within a matrix,or being adhered to a matrix.

Much like their antimicrobial activity in the body, the antimicrobialactivity of the polymers of the present invention may further beharnessed for the preservation of food ingredients and products.

Hence, according to yet another aspect of the present invention there isprovided a food preservative comprising an effective amount of thepolymer of the present invention as described herein.

The polymer(s) may be incorporated into the food product as one of itsingredients either per se, or with an edible carrier.

The polymers of the present invention have been shown to have high andselecting affinity towards membranes of microorganisms as demonstratedin the Examples section that follows. This attribute is one of the mainelements which contribute to the effective and efficacious activity ofthe polymers when utilized as an antimicrobial agent. When the polymeris coupled with a labeling agent, this membrane binding attribute can befurther employed to label colonies and proliferation sites ofmicroorganisms, especially microbial growth loci in a host in vivo.

Hence, according to another aspect of the present invention there isprovided an imaging probe for detecting a pathogenic microorganism, theimaging probe comprising a polymer as defined and described hereinabove,whereas the polymer further includes at least one labeling agent, asdefined hereinabove, attached thereto. When released to the environment,these polymers, having a labeling agent attached thereto will bind tothe membrane of cell of microorganisms and therefore attach the labelingagent to the cells of the microorganism.

As used herein, the term “chromophore” refers to a chemical moiety that,when attached to another molecule, renders the latter colored and thusvisible when various spectrophotometric measurements are applied.

The phrase “fluorescent compound” refers to a compound that emits lightat a specific wavelength during exposure to radiation from an externalsource.

The phrase “phosphorescent compound” refers to a compound emitting lightwithout appreciable heat or external excitation as by slow oxidation ofphosphorous.

A heavy metal cluster can be for example a cluster of gold atoms used,for example, for labeling in electron microscopy techniques.

According to preferred embodiments of the present invention, one or morelabeling agents may be attached to the polymer at any substitutableposition, as in the case of an active agent discussed above. Examples ofsuch substitutable positions are, without limitation, a side chain ofany one or more of the amino acid residues in the polymer, any one ofthe linking moieties of the polymer, any one of the N- and C-termini ofthe polymer and any one or more of the hydrophobic moiety residues inthe polymer.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Examples

Reference is now made to the following examples, which together with theabove descriptions; illustrate the invention in a non limiting fashion.

Materials and Experimental Methods Chemical Syntheses

Materials:

Lysine having Fmoc ((9H-fluoren-9-yl)methyl carbonate) protection on itsmain-chain amine group and Boc (tert-butyl carbonate) protection on itsside-chain amine group was purchased from Applied Biosystems and fromNovaBiochem.

ω-amino fatty acids such as 4-amino-butyric acid, 8-amino-caprylic acidand 12-amino-lauric acid having Fmoc protection of the amine group werepurchased from Sigma-Aldrich/NovaBiochem.

All other solvents and reagents used were purchased fromSigma-Aldrich/NovaBiochem/Applied Biosystems/J.T.Baker and were usedwithout further purification.

Preparation of Libraries of Antimicrobial Polymers—General Procedure:

The polymers according to the present invention were prepared by a solidphase method and were purified to chromatographic homogeneity accordingto methodologies described in the art (Feder, R. et al. (2000) J. Biol.Chem. 275, 4230-4238). Briefly, the polymers were synthesized byapplying the Fmoc active ester chemistry on a fully automated,programmable peptide synthesizer (Applied Biosystems 433A). Aftercleavage from the resin, the crude polymers were extracted with 30%acetonitrile in water and purified to obtain a chromatographichomogeneity greater than 95%, as determined by HPLC (Alliance Waters).

HPLC chromatograms were performed on C18 columns (Vydak, 250 mm×4.6 or10 mm) using a linear gradient of acetonitrile in water (1% per minute),both solvents contained 0.1% trifluoroacetic acid. The purified polymerswere subjected to mass spectrometry (ZQ Waters) to confirm theircomposition and stored as a lyophilized powder at −20° C. Prior to beingtested, fresh solutions were prepared in water, mixed by vortex,solubilized by ultrasound, centrifuged and then diluted in theappropriate medium.

In order to estimate the hydrophobicity of each polymer, the polymer waseluted with a linear gradient of acetonitrile (1% per minute) on an HPLCreversed-phase C18 column, and the percent of acetonitrile at which thepolymer was eluted was used for hydrophobicity estimation (see, “ACN(%)” in Table 3 below).

Exemplary building units which were utilized in the synthesis describedabove are presented in Scheme 1 below and include: lysine and anω-amino-fatty acid having m carbon atoms (Compound I).

Synthesis of exemplary polymers according to the present invention,which are comprised of lysine and Compound I, was performed by adding anFmoc/Boc-protected lysine and an Fmoc-protected Compound I separatelyand sequentially to the resin according to conventional peptide solidphase synthesis protocols.

In Vitro Studies:

Bacterial Strains and Sample Preparation:

Antibacterial activity was determined using the following strains,cultured in LB medium (10 grams/liter trypton, 5 grams/liter yeastextract, 5 grams/liter NaCl, pH 7.4): Escherichia coli (ATCC (AmericanType Culture Collection) 35218); methicilin resistant Staphylococcusaureus (CI (clinical isolate) 15903); Bacillus cereus (ATCC 11778); andPseudomonas aeruginosa (ATCC 9027).

Minimal Inhibitory Concentration (MIC) Measurements:

Minimal inhibitory concentrations (MICs) were determined bymicrodilution susceptibility testing in 96-well plates using inocula of10⁶ bacteria per ml.

Cell populations were evaluated by optical density measurements at 600nm and were calibrated against a set of standards. Hundred (100) μl of abacterial suspension were added to 100 μl of culture medium (control) orto 100 μl of culture medium containing various polymer concentrations in2-fold serial dilutions. Inhibition of proliferation was determined byoptical density measurements after an incubation period of 24 hours at37° C.

Alternatively, MICs were determined using the microbroth dilution assayrecommended by the Clinical and Laboratory Standards Institute (CLSI)using two-fold serial dilutions in cation-adjusted Mueller-Hinton broth(CAMHB).

Clinical bacterial isolates were obtained from Tel Aviv Sourasky MedicalCenter, Israel. Bactericidal kinetics was assessed using the drop platemethod [see, for example, Chen et al., Journal of MicrobiologicalMethods, November; 55(2):475-9, 2003; and Skerman V. B. D., 1969,Abstracts of Microbiological Methods, p. 143-161, Wiley-Interscience,New York]. Statistical data for each experiment were obtained from atleast two independent assays performed in duplicates.

The Effect of Physical Parameters (Charge and Hydrophobicity) onAntimicrobial Activity:

A library of polymers was prepared to sample the effect of increasedcharge and hydrophobicity on the antimicrobial activity. The charge wasserially sampled by increasing the number of the ω-amino-fattyacid-lysine conjugates from 1 to 7. The hydrophobicity was seriallysampled by increasing the number of the carbon atoms of the ω-aminofatty acid (4, 8 and 12). The polymers in each series were tested fortheir antimicrobial activity, as described hereinabove.

Development of Antimicrobial-Resistance in Bacteria:

The possible development of resistance to the antimicrobial activity ofthe polymers of the present invention by bacteria, as compared withknown resistance-inducing classical antibiotic agents, gentamycin,tetracycline and ciprofloxacin, which served as controls for thedevelopment of antibiotic-resistant bacterial strains, was studied.

Bacteria samples (E. coli strain ATCC 35218) at the exponential phase ofgrowth were exposed to an antimicrobial agent for MIC determination asdescribed above. Following incubation overnight, bacteria were harvestedfrom wells that displayed near 50% growth inhibition, washed and dilutedin fresh medium, grown overnight, and subjected again to MICdetermination for up to 15 iterations (15 days). For each compoundtested, the 0D₆₂₀ of one half the MIC well from the previous MIC assaywas diluted to yield 5×10⁵ cells/ml in LB (according to a calibrationcurve) and was used again for MIC determination in subsequentgenerations.

In parallel, MIC evolution in these subcultures was comparedconcomitantly with each new generation, using bacteria harvested fromcontrol wells (wells cultured without a polymer) from the previousgeneration. The relative MIC was calculated for each experiment from theratio of MIC obtained for a given subculture to that obtained forfirst-time exposure.

Kinetic Studies:

The kinetic assays were performed in test tubes, in a final volume of 1ml, as follows: 100 μl of a suspension containing bacteria at 2-4×10⁷colony forming units (CFUs)/ml in culture medium were added to 0.9 ml ofculture medium or culture medium containing various polymerconcentrations (0, 3 and 6 multiples of the MIC value). After 0, 30, 60,90, 120 and 360 minutes of exposure to the polymer at 37° C. whileshaking, cultures were subjected to serial 10-fold dilutions (up to10⁻⁶) by adding 50 μl of sample to 450 μl saline (0.9% NaCl). Colonyforming units (CFUs) were determined using the drop plate method (3drops, 20 μl each, onto LB-agar plates, as described in Yaron, S. et al.(2003), Peptides 24, 1815-1821). CFUs were counted after plateincubation for 16-24 hours at 37° C. Statistical data for each of theseexperiments were obtained from at least two independent assays performedin duplicates.

Antimicrobial Activity at Enhanced Outer-Membrane PermeabilityConditions:

The outer membrane permeability of gram-negative bacteria, namely E.coli or P. aeruginosa, was enhanced by treating bacterial cultures withEDTA (ethylenediaminetetraacetic acid) according to the followingprocedure: 1 M EDTA solution in water (pH=8.3) was diluted in LB mediumto obtain a 4 mM concentration and the diluted solution was used forpolymer dissolution. Bacteria were grown overnight in LB medium, and 100μl fractions containing 10⁶ bacteria per ml were added to 100 μl of EDTAculture medium or to EDTA culture medium containing various polymerconcentrations (2-fold serial dilutions) in 96-well plates. Growthinhibition was determined against gram-negative bacteria as describedabove.

Susceptibility to Plasma Proteases:

The susceptibility of the polymers of the present invention toproteolytic digestion was assessed by determining the antibacterialactivity after exposure to human plasma as follows: 250 μl of polymersolutions in saline (0.9% NaCl) at a concentration of 16 multiples ofthe MIC value were pre-incubated with 50% (v/v) human plasma in culturemedium at 37° C. After incubation periods of 3, 6, and 18 hours, thepolymer solutions were subjected to 2-fold serial dilutions in LB mediumin 96-well plates. The susceptibility of the polymers of the presentinvention to enzymatic cleavage was assessed by pre-incubating fourexemplary polymers according to the present invention, C₁₂K(NC₈K)₅NH₂,K(NC₁₂K)₃NH₂, C₁₂KNC₁₂KNH₂, and C₁₂KKNC₁₂KNH₂, and a 16-residuesdermaseptin S4 derivative (S4₁₆, an exemplary AMP which served as thecontrol) (SEQ ID NOs: 43, 60, 54, 78 and 98 respectively), in humanplasma (50%) for various time periods. The antibacterial activity wasthereafter determined against E. coli and S. aureus, as described above.In parallel, antibacterial activity was also determined in culturemedium conditions in the absence of plasma (referred to as 0 hours ofpre-incubation in the experimental results section below). Statisticaldata was obtained from at least two independent experiments performed induplicates.

Hemolysis Assays:

The polymer's membranolytic potential was determined against human redblood cells (RBC) in phosphate buffer solution (PBS). Human bloodsamples were rinsed three times in PBS by centrifugation for 2 minutesat 200×g, and re-suspended in PBS at 5% hematocrit. A 50 μl-fractions ofa suspension containing 2.5×10⁸ RBC were added to test tubes containing200 μl of polymer solutions (2-fold serial dilutions in PBS), PBS alone(for base-line values), or distilled water (for 100% hemolysis). After 3hours incubation at 37° C. under agitation, samples were centrifuged,and hemolytic activity was determined as a function of hemoglobinleakage by measuring absorbance at 405 nm of 200 μl aliquots of thesupernatants.

Alternatively, a 10% hematocrit was used and hemolysis was determinedafter one hour incubation. Hemolytic activity was determined accordingto Antibacterial Peptides Protocols as presented by Tossi, A. et al. inMethods Mol. Biol., 1997, 78, pp. 133-150.

Circular Dichroism (CD):

CD spectra in millidegrees were measured with an Aviv model 202 CDspectrometer (Aviv Associates, Lakewood, N.J.) using a 0.01 cmrectangular QS Hellma cuvette at 25° C. (controlled by thermoelectricPeltier elements with an accuracy of 0.1° C.). Polymer samples weredissolved in either PBS, 20% (v/v) trifluoroethanol/water or titrated inPBS containing POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine)and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol)) ina 3:1 ratio and concentration of up to 2 mM, to thereby obtainliposomes. CD spectra of the polymers were scanned at a concentration of100 μM as determined by UV using standard curves of known concentrationsfor each polymer. The CD of the N-terminus acylated S4 dermaseptinderivative NC₁₂K₄54 (1-14) (SEQ ID NO: 101) (Mor, A. et al. (1994), J.Biol. Chem. 269(50): 31635-41), an exemplary AMP, was measured under thesame conditions and was used as a reference compound in the CD studies.The CD data presented herein represent an average of three separaterecordings values.

Surface Plasmon Resonance Assay:

Binding to model bilayer membranes was studied by surface plasmonresonance (SPR) using a BIAcore 2000 biosensor system. Liposomescomposed of phospholipids mimicking bacterial plasma membrane (POPC:POPGin a 3:1 ratio) were immobilized on the sensor surface and polymersolutions were continuously flowed over the membrane. The curve ofresonance signal as a function of time displays the progress of theinteraction between the analyzed polymer and the immobilizedphospholipid membrane. The affinity of the interaction was calculatedfrom analysis of the resulting curves as detailed in Gaidukov, L. et al.(2003), Biochemistry 42, 12866-12874. Briefly, the association anddissociation curves (binding rates) were analyzed at five doses (0.21,0.42, 0.84, 1.67, 3.35 μg), and the K_(app) (the resulting bindingconstant) was calculated assuming a 2-step model).

Lipopolysaccharide Binding Assay:

In order to explore the mechanism by which the polymers of the presentinvention exert the anti-bacterial activity, the targeting of thepolymers to the bacterial membrane was tested. More specifically, thebinding affinity of the positively charged polymers to the negativelycharged lipopolysaccharides (LPS) present on the membrane ofgram-negative bacteria was tested.

Thus, binding assays of the polymers of the present invention to LPSwere carried out with SPR technology using the optical biosensor systemBIAcore 2000 (BIAcore). A mixture of 50 μM of the polymer samples in PBSand 100 μg/ml LPS was incubated for 30 minutes at room temperature. Thebinding assay was performed by injecting 10 μl of the mixture at a flowrate of 10 μl per minute at 25° C. over a POPC:POPG (3:1) bilayer spreadon an L1 sensor chip. 100 μg/ml LPS without a polymer sample wereinjected as a blank of LPS binding to the membrane and 50 μM of apolymer sample was injected to determine the polymer binding to membranewithout LPS.

DNA Binding Assay:

Binding of the polymers of the present invention to nucleic acids wasstudied by assessing their ability to retard migration of DNA plasmidsduring gel electrophoresis in a 1% agarose gel. DNA-retardationexperiments were performed by mixing 200 nanograms of the plasmid DNA(pUC19, 2683 base pairs) with increasing amounts of various polymers ina final volume of 20 μl doubly distilled water (DDW). The reactionmixtures were incubated at room temperature for 30 minutes.Subsequently, 2 μl of loading dye (20% Ficoll 400, 0.1 M EDTA, 0.25%bromophenol blue and 1% sodium dodecyl sulfate) were added and analiquot of 20 μl was applied to 1% agarose gel electrophoresis in TAEbuffer (0.02 M Tris base, 0.01 M glacial acetic acid, 0.5 mM EDTA,pH=8.5) containing ethidium bromide (0.25 μg/ml). The plasmid used inthis experiment was isolated by the Wizard® Plus SV Minipreps DNAPurification System (Promega).

Saliva Microbicidal Assays:

Antimicrobial activity of polymers of the present invention against themélange of microorganisms in the saliva of healthy human volunteers wasstudied by mixing fresh human saliva with the polymers or IB-367 (SEQ IDNO: 97) (both dissolved in 10 mM sodium acetate buffer set at pH 5 to afinal concentration of 100 μM) at a 1:1 ratio. A solution of the salivawith no anti-bacterial agent served as a control. IB-367 (SEQ ID NO: 97)is a positively charged protegrin peptide with known in-vitro andin-vivo activities against the microflora associated with human oralmucositis (Loury, D. et al., 1999, Oral Surg Oral Med Oral Pathol OralRadiol Endod 87(5): 544-51.). Each of the solutions was spread over a LAplate, and the plated saliva samples were incubated overnight at 37° C.without aeration. The colonies were enumerated and counted to determinethe microbicidal effect of the drug. The values of viable colony formingunits (CFU) were determined as described above.

Anti-Malarial Assays:

The investigation of the anti-malarial activity of the polymers of thepresent invention was performed by screening part of the library of thepolymers presented hereinbelow in Table 3, for anti-malarial andhemolytic activities as well as for their toxic activities againstmammalian cells in culture.

Parasite cultivation: Different strains of P. falciparum were cultivatedas described by Kutner and co workers [Kutner, S., Breuer, W. V.,Ginsburg, H., Aley, S. B., and Cabantchik, Z. I. (1985) J. Cell.Physiol. 125, 521-527] using human red blood cells (RBC). The cultureswere synchronized by the sorbitol method [Lambros, C. J., andVanderberg, J. P. (1979) J. Parasitol. 65, 418-420] and infected cellswere enriched from culture by Percoll-alanine gradient centrifugation[Kutner, S., Breuer, W. V., Ginsburg, H., Aley, S. B., and Cabantchik,Z. I. (1985) J. Cell. Physiol. 125, 521-527].

Determination of IC₅₀: Synchronized cultures at the ring stage werecultured at 1% hematocrit and 2% parasitemia in the presence ofincreasing concentrations of the tested polymers. After 18 hours ofincubation parasite viability was determined by [³H]hypoxanthine (Hx)uptake (final concentration was 2 μCi/ml) during 6 hours and compared tocontrols (without the polymers). The 50% inhibitory concentration (IC₅₀)was determined by nonlinear regression fitting of the data using thecommercially available software suite Sigmaplot™.

Time- and stage-dependence action of the polymers: Anti-malarial drugsare known to exert their action differentially on different stages ofparasite development. They also need a minimal time of interaction withthe parasite in order to inhibit its growth. Therefore, cultures at thering stage were seeded in 24-well plate at 1% hematocrit, 2% parasitemiain plate medium (growth medium without hypoxanthine, 10 mM NaHCO3 and 7%heat inactivated human plasma). Tested polymers were added at differentconcentrations immediately and removed after 6, 24 and 48 hours.Cultures without polymers were left to mature to the trophozoite stageand dosed with compounds for 6 and 24 hours. Two μCi of Hx per well wereadded to all cells after 30 hours from the onset of the experiment andthe cells were harvested after 24 hours.

Effect of the polymers on mammalian cells in culture: MDCK (cell linefrom dog kidney) epithelial cells were grown to confluence (about 3 daysin culture). Parallel cultures were grown with different concentrationsof the tested polymers. Thereafter 10 μl of Alamar blue was added andfluorescence was measured after 3.5 hours. For a positive control, 10 μMof cycloheximide were added to control samples at the beginning ofcultivation.

In-Vivo Studies:

Activity Assay:

In-vivo prevention of E. coli-induced mortality using acute peritonitisand sepsis model experiments were conducted using female neutropenic ICR(Institute for Cancer Research) mice (weighing 25 to 27 grams each).

A bacterial inoculum was prepared on brain heart infusion (BHI) broth(Becton-Dickinson) with agitation (180 rpm) at 37° C. for 5 to 6 hours.Cells were harvested when the culture reached an optical density of 0.8OD₆₂₀ and re-suspended in sterile BHI broth. The number of viable cellswas verified by plating serial dilutions of the injected inocula ontoBHI agar plates.

Infection was induced by intraperitoneal injection of 2.5×10⁶ or 5×10⁶colony forming units (CFU) of E. coli in the logarithmic growth phase(CI 3504, isolated from a patient with peritonitis and bacteremia) in0.5 ml of culture media to groups of 10 mice each. One hour afterinfection, mice were intraperitoneally injected with 0.5 ml of vehicle(PBS control), one 4 mg/kg body weight dose of an exemplary polymer,C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), or 4 doses injection (after 1, 6, 20,and 28 hours) of 2 mg/kg body weight of imipenem (an antibiotic drugwhich belongs to the carbapenems family and used to treat severe or veryresistant infections). Mice were monitored for survival over 6 daysperiod after infection.

Toxicity:

Acute toxicity was examined after intraperitoneal injection ofC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplary polymer according to thepresent invention, to groups of 12 ICR mice. Each mouse was injectedwith a 0.5 ml solution of freshly prepared C₁₂K(NC₈K)₇NH₂ (SEQ ID NO:45) in PBS. The doses of polymer administered per mouse were 0 μg (blankcontrol), 100 μg, 250 μg, and 500 μg (corresponding to 0 mg/kg, 4 mg/kg,10 mg/kg, and 20 mg/kg body weight). Animals were directly inspected foradverse effects for 4 hour, and mortality was monitored for 7 daysthereafter.

EXPERIMENTAL RESULTS Preparation of Libraries of Polymers

Several representative series of polymers according to the presentinvention, which are substantially comprised of a plurality of lysineresidues and ω-amino-fatty acid residues and fatty acid residues ashydrophobic moieties, were prepared according to the general proceduredescribed above, and are presented in Table 3 below.

These exemplary polymers are referred to in this section according tothe following formula:

T[NC_(i)K]_(j)G

In this formula, NC_(i) denotes an ω-amino-fatty acid residue (anexemplary hydrophobic moiety according to the present invention,represented by D₁ . . . Dn in the general formula I described herein),whereby i denotes the number of carbon atoms in the fatty acid residue;K denotes a lysine residue (an exemplary amino acid residue according tothe present invention, denoted as A₁ . . . An in the general Formula Idescribed herein, such that [NC_(i)K] denotes a residue of anω-amino-fatty acid-lysine conjugate (denoted as [A₁-Z₁-D₁] . . .[An-Zn-Dn] in the general Formula I described herein); j denotes thenumber of the repeating units of a specific conjugate in the polymer(corresponding to n in the general Formula I described herein); and Tand G each independently denotes either a hydrogen (no denotation), alysine residue (denoted K), an ω-amino-fatty acid residue (denotedNC_(i)), a fatty acid residue (denoted C_(i)), an ω-amino-fattyacid-lysine conjugate residue (denoted NC_(i)K), afluorenylmethyloxycarbonyl residue (denoted Fmoc), a benzyl residue(denoted Bz), a cholate residue (denoted Chl), an amine group (typicallyforming an amide at the C-terminus and denoted NH₂), and free acidresidue (for the C-terminus no denotation), an alcohol residue, and anycombination thereof (all corresponding to X and Y in the general formulaI described herein).

Thus, for example, a polymer according to the present invention which isreferred to herein as NC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 52), corresponds to apolymer having the general formula I described hereinabove, wherein: Xis a residue of a conjugate of an ω-amino-fatty acid having 12 carbonatoms (12-amino-lauric acid) and lysine; n is 6; A₁ . . . A₆ are each alysine residue; D₁ . . . D₇ are all residues of an ω-amino-fatty acidhaving 8 carbon atoms (8-amino-caprylic acid); Z₁ . . . Z₇ and W₀-W₇ areall peptide bonds; and Y is an amine. For clarity, the chemicalstructure of NC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 52) is presented in Scheme 2below:

Minimal Inhibitory Concentration Measurements:

The polymers in each series were tested for various antimicrobialactivities, as described hereinabove. The obtained results are presentedin Table 3 below, wherein:

“Q” represents the overall molecular charge at physiological pH (column3 in Table 3);

“ACN (%)” represents the percent of acetonitrile in the HPLC-RP gradientmobile phase at which the polymer was eluted and which corresponds tothe estimated hydrophobicity of the polymer (column 4 in Table 3);

“LC50” represents the lytic concentration of each tested polymer in μMobtained by the membranolytic potential determination experiment ofhemolysis of human red blood cells measured as described hereinabove(column 5 in Table 3);

“MIC E.c.” represents the minimal inhibitory concentration of eachtested polymer in μM for E. coli, measured as described hereinabove inthe antibacterial activity assay (column 6 in Table 3);

“MIC EDTA E.c.” represents the minimal inhibitory concentration of eachtested polymer in μM for E. coli culture in the presence of 2 mM EDTA,measured as described hereinabove for the enhanced outer-membranepermeability assay (column 7 in Table 3);

“MIC P.a.” represents the minimal inhibitory concentration of eachtested polymer in μM for P. aeruginosa, measured as describedhereinabove for the antibacterial activity assay (column 8 in Table 3);

“MIC MR S.a.” represents the minimal inhibitory concentration of eachtested polymer in μM for methicilin-resistant S. aureus, measured asdescribed hereinabove for the antibacterial activity assay ofantibiotic-resistant bacteria (column 9 in Table 3);

“MIC B.c.” represents the minimal inhibitory concentration of eachtested polymer in μM for Bacillus cereus, measured as describedhereinabove for the antibacterial activity assay (column 10 in Table 3);and

ND denotes “not determined”.

Some values are presented with ±standard deviations from the mean.

“Orn” and “Arg” in entries 84 and 85 denote ornithine and arginine aminoacid residues respectively.

Entries 96 to 99 present activity data of four control antimicrobialpeptides, namely MSI-78 (SEQ ID NO: 96), a magainin derivative; IB-367(SEQ ID NO: 97), a protegrin derivative; K₄S₄(1-16) (SEQ ID NO: 98), adermaseptin derivative; and LL37 (SEQ ID NO: 99), a cathelicidinderivative.

Entries 100 to 103 present activity data of four control antibioticagents, namely Ciprofloxacin, Imipenem, Tetracycline and Rifampin.

TABLE 3 MIC MIC Entry ACN MIC EDTA MIC MR MIC No. SEQ Polymer Q (%) LC50E.c. E.c. P.a. S.a. B.c. 1 1 C₄KNC₄KNH₂ 2 19.6 >100 >50 ND >50 >50 >50 22 C₄K(NC₄K)₂NH₂ 3 21.8 >100 >50 ND >50 >50 >50 3 3 C₄K(NC₄K)₃NH₂ 421.5 >100 >50 ND >50 >50 >50 4 4 C₄K(NC₄K)₄NH₂ 5 22 >100 >50ND >50 >50 >50 5 5 C₄K(NC₄K)₅NH₂ 6 22.9 >100 >50 ND >50 >50 >50 6 6C₄K(NC₄K)₆NH₂ 7 23.5 >100 >50 ND >50 >50 >50 7 7 C₄K(NC₄K)₇NH₂ 824.3 >100 >50 ND >50 >50 >50 8 8 KNC₄KNH₂ 3 0 ND >50 ND >50 >50 >50 9 9K(NC₄K)₂NH₂ 4 9 ND >50 ND >50 >50 >50 10 10 K(NC₄K)₃NH₂ 5 18.8 ND >50ND >50 >50 >50 11 11 K(NC₄K)₄NH₂ 6 20.1 ND >50 ND >50 >50 >50 12 12K(NC₄K)₅NH₂ 7 20.8 ND >50 ND >50 >50 >50 13 13 K(NC₄K)₆NH₂ 8 21.7 ND >50ND >50 >50 >50 14 14 K(NC₄K)₇NH₂ 9 22.2 ND >50 ND >50 >50 >50 15 15C₁₂K(NC₄K)₁NH₂ 2 50.2 ND >50 ND >50 >50 >50 16 16 C₁₂K(NC₄K)₂NH₂ 3 47.6ND >50 ND >50 >50 >50 17 17 C₁₂K(NC₄K)₃NH₂ 4 46.4 ND >50 ND >50 >50 >5018 18 C₁₂K(NC₄K)₄NH₂ 5 45.4 ND 50 ND >50 >50 >50 19 19 C₁₂K(NC₄K)₅NH₂ 645.8 ND 12.5 6.3 >50 >50 >50 20 20 C₁₂K(NC₄K)₆NH₂ 7 45.1 ND  9.4 ± 3.14.7 ± 2.2 >50 >50 >50 21 21 C₁₂K(NC₄K)₇NH₂ 8 45.2 >100  9.4 ± 3.13.1 >50 >50 >50 22 22 NC₁₂K(NC₄K)₅NH₂ 7 29.3 ND >50 ND >50 >50 >50 23 23NC₁₂K(NC₄K)₆NH₂ 8 29.8 ND >50 ND >50 >50 >50 24 24 NC₁₂K(NC₄K)₇NH₂ 930.2 ND >50 ND >50 >50 >50 25 25 C₈KNC₈KNH₂ 2 38.9 >100 >50ND >50 >50 >50 26 26 C₈K(NC₈K)₂NH₂ 3 36 >100 >50 ND >50 >50 >50 27 27C₈K(NC₈K)₃NH₂ 4 39.5 >100 >50 ND >50 >50 >50 28 28 C₈K(NC₈K)₄NH₂ 540.5 >100 >50 ND >50 >50 >50 29 29 C₈K(NC₈K)₅NH₂ 6 40.8 >100 253.1 >50 >50 >50 30 30 C₈K(NC₈K)₆NH₂ 7 40.3 >100 25 ND >50 >50 >50 31 31C₈K(NC₈K)₇NH₂ 8 40.3 >100 12.5 ND >50 >50 >50 32 32 KNC₈KNH₂ 3 21.6ND >50 ND >50 >50 >50 33 33 K(NC₈K)₂NH₂ 4 27.3 ND >50 ND >50 >50 >50 3434 K(NC₈K)₃NH₂ 5 30 ND >50 ND >50 >50 >50 35 35 K(NC₈K)₄NH₂ 6 31.7ND >50 ND >50 >50 >50 36 36 K(NC₈K)₅NH₂ 7 33.1 ND >50 >50 >50 >50 >50 3737 K(NC₈K)₆NH₂ 8 33.4 ND >50 >50 >50 >50 >50 38 38 K(NC₈K)₇NH₂ 9 34.2ND >50 37 ± 5  >50 >50 >50 39 39 C₁₂KNC₈KNH₂ 2 50.9 ND >50ND >50 >50 >50 40 40 C₁₂K(NC₈K)₂NH₂ 3 48 ND >50 ND >50 >50 >50 41 41C₁₂K(NC₈K)₃NH₂ 4 46 ND >50 ND >50 >50 >50 42 42 C₁₂K(NC₈K)₄NH₂ 5 49 ND25 4.7 ± 2.2 >50 37.5 ± 18 50 43 43 C₁₂K(NC₈K)₅NH₂ 6 49.7 >100 3.1 0.450 50 12.5 44 44 C₁₂K(NC₈K)₆NH₂ 7 50 >100 3.1 0.8 50 50 12.5 45 45C₁₂K(NC₈K)₇NH₂ 8 47.5 >100 3.1 ND 6.2 50 12.5 46 46 NC₁₂KNC₈KNH₂ 3 29.6ND >50 ND >50 >50 >50 47 47 NC₁₂K(NC₈K)₂NH₂ 4 35 ND >50 ND >50 >50 >5048 48 NC₁₂K(NC₈K)₃NH₂ 5 33.7 ND >50 ND >50 >50 >50 49 49 NC₁₂K(NC₈K)₄NH₂6 36.2 ND >50 12.5 >50 >50 >50 50 50 NC₁₂K(NC₈K)₅NH₂ 7 36.6 ND 506.3 >50 >50 >50 51 51 NC₁₂K(NC₈K)₆NH₂ 8 37 ND 25 6.3 >50 >50 >50 52 52NC₁₂K(NC₈K)₇NH₂ 9 36.9 ND 12.5 ND 50 >50 >50 53 53 C₁₂KK(NC₈K)₄NH₂ 647 >100 6.3 ND 50 37.5 ± 18 25 54 54 C₁₂KNC₁₂KNH₂ 2 59  45 ± 12 20.8 ±7.2 ND 50  18.8 ± 7.2 >50 55 55 C₁₂K(NC₁₂K)₂NH₂ 3 52.9 ND >50 37.5 ±18   >50 >50 >50 56 56 C₁₂K(NC₁₂K)₃NH₂ 4 53.5 ND >50 >50 >50 >50 >50 5757 C₁₂K(NC₁₂K)₄NH₂ 5 53.4 ND >50 >50 >50 >50 >50 58 58 KNC₁₂KNH₂ 332 >100 >50 >50 >50 >50 >50 59 59 K(NC₁₂K)₂NH₂ 440 >100 >50 >50 >50 >50 >50 60 60 K(NC₁₂K)₃NH₂ 5 44 >100 12.5 3.125 >50 >50 61 61 K(NC₁₂K)₄NH₂ 6 46  6.5 ± 3.5 25 2.3 ± 1.1 >50 >50 >5062 62 K(NC₁₂K)₅NH₂ 7 47 ND >50 3.1 ND >50 >50 63 63 K(NC₁₂K)₆NH₂ 8 48ND >50 3.1 ND ND >50 64 64 K(NC₁₂K)₇NH₂ 9 50 ND >50 6.3 ND ND >50 65 65(NC₁₂K)₂NH₂ 3 38.8 >100 >50 >50 >50 >50 >50 66 66 (NC₁₂K)₃NH₂ 444.3 >100 25 6.3 50 >50 >50 67 67 (NC₁₂K)₄NH₂ 5 46.8   4 ± 1.4 >506.3 >50 >50 >50 68 68 (NC₁₂K)₅NH₂ 6 47.8 ND >50 12.5 >50 >50 >50 69 69(NC₁₂K)₆NH₂ 7 49 ND >50 3.1 ND >50 ND 70 70 (NC₁₂K)₇NH₂ 8 50 ND >50 12.5ND ND ND 71 71 (NC₁₂K)₈NH₂ 9 51 ND >50 1.6 ND ND ND 72 72 KKNC₁₂KNH₂ 430.9 >100 >50 ND >50 >50 >50 73 73 (KNC₁₂K)₂NH₂ 5 38.1 >100 >50 ND50 >50 >50 74 74 K(KNC₁₂K)₂NH₂ 6 37.3 >100 >50 ND 25 >50 >50 75 75C₈KKNC₁₂KNH₂ 3 40.3 >100 >50 ND >50 >50 >50 76 76 C₈(KNC₁₂K)₂NH₂ 445 >100 25 ND 12.5 >50 3.1 77 77 C₈K(KNC₁₂K)₂NH₂ 5 42.6 >100 37.5 ± 18 ND 12.5 >50 6.3 78 78 C₁₂KKNC₁₂KNH₂ 3 54 28.5 ± 9.2 18.8 ± 8.8 9.4 ± 4.425 3.1 3.1 79 79 C₁₂(KNC₁₂K)₂NH₂ 4 53.3 16.5 ± 6.4 3.1 ND 3.1 1.6 3.1 8080 C₁₂K(KNC₁₂K)₂NH₂ 5 51 88 ± 3 3.1 ND 3.1 12.5 3.1 81 81 NC₁₂KKNC₁₂KNH₂4 38.9 >100 >50 ND >50 >50 >50 82 82 NC₁₂(KNC₁₂K)₂NH₂ 5 38.5 >100 50 ND12.5 >50 3.1 83 83 NC₁₂K(KNC₁₂K)₂NH₂ 6 38.6 >100 25 ND 25 >50 6.3 84 84C₁₂OrnNC₁₂OrnNH₂ 2 53.8 24 ± 6 10.4 ± 3.6 ND 25 12.5 16.7 ± 7.2   85 85C₁₂ArgNC₁₂ArgNH₂ 2 57.1 9.5 ± 1    42 ± 14.4 ND >50 12.5 42 ± 14.4 86 86C₁₂KNC₁₂K 1 56.9 >100 >50 ND >50 >50 >50 87 87 C₁₂K(NC₁₂K)₂ 2 56.4ND >50 ND >50   31.3 ± 26.5 50 88 88 C₁₂K(NC₁₂K)₃ 3 54.6 ND >50ND >50 >50 >50 89 89 KNC₁₂K 2 33.2 >100 >50 ND >50 >50 >50 90 90K(NC₁₂K)₂ 3 36.4 >100 >50 ND >50 >50 >50 91 91 K(NC₁₂K)₃ 4 42.8 >100 25ND 50 >50 >50 92 92 (NC₁₂K)₂ 2 38.7 >100 >50 ND >50 >50 >50 93 93(NC₁₂K)₃ 3 43.8 >100 50 ND >50 50 >50 94 94 (NC₁₂K)₄ 4 45.8 ND 50ND >50 >50 >50 95 95 FmocK(NC₁₂K)₂ 2 41 ND >50 ND >50 6.3 12.5 96 96MSI-78 10 44   45 50 ND 3.1 >50 37.5 97 97 IB-367 5 45   7 3.1 ND 12.53.1 ND 98 98 K₄S₄(1-16) 6 47   10 3.1 ND 6.3 6.3 3.1 99 99 LL37 11 61  8 50 ND 12.5 ND 37.5 100 Ciprofloxacin ND ND ND 0.05 ND 0.3 >50 0.3101 Imipenem ND ND ND 0.6 ND 16.4 >50 <0.03 102 Tetracycline ND ND ND1.8 ND >50 0.4 0.07 103 Rifampin ND ND ND 7.7 ND 15.2 0.006 0.09

The Effect of Physical Parameters (Charge and Hydrophobicity) onAntimicrobial Activity:

Charge and hydrophobicity may be viewed as two conflicting physicalcharacteristics of a molecule: charge facilitates dissolution of acompound in aqueous media by interacting with the polar water molecules,while hydrophobicity, which typically corresponds to the number andlength of non-polar hydrocarbon moieties, hinders dissolution.Optimization of these physical characteristics is crucial in thedevelopment of drugs in general and antimicrobial agents in particular,as these characteristics affect pharmaceutically important traits suchas membrane permeability and transport in and across biological systems.

Therefore, the library of polymers prepared to study the effect ofserial increases in charge and hydrophobicity properties was measuredfor its antimicrobial activity against two gram-negative bacteria: E.coli (results are presented in column 6 of Table 3 hereinabove) and P.aeruginosa (results are presented in column 8 of Table 3), and twogram-positive bacteria: methicilin-resistant S. aureus (results arepresented in column 9 of Table 3) and Bacillus cereus (results arepresented in column 10 of Table 3).

A serial increase in positive charge was achieved by preparing polymerswith serial elongation of the chain with respect to the number of lysineresidues. Serial increases in hydrophobicity was achieved by preparingpolymers with serial rising of the number of fatty acid residues (as arepresentative hydrophobic moiety) and/or with serial rising of thenumber of carbon atoms in each fatty acid residue. Serial increases inboth positive charge and hydrophobicity were achieved by preparingpolymers with serial rising of the number of lysine-amino fatty acidconjugates.

As can be seen in Table 3, increasing the hydrophobicity of the polymersby increasing the number of the carbon atoms in the fatty acid residuefrom 4 to 12, via 8 carbon atoms, was found to affect the antimicrobialactivity of the polymers. Series of polymers in which the repeatinghydrophobic moiety was a 4-amino-butyric acid (see, entries 1-24 inTable 3) was compared to a series in which the repeating hydrophobicmoiety was an 8-amino-caprylic acid (see, entries 25-53 in Table 3) andto a series in which the repeating hydrophobic moiety was a12-amino-lauric acid (see, entries 54-95 in Table 3). The results,presented in Table 3, indicated that polymers in which the repeatinghydrophobic moiety was a 4-amino-butyric acid (see, entries 1-14 inTable 3) and a 8-amino-caprylic acid (see, entries 25-38 in Table 3),generally did not show significant antimicrobial activity up to thehighest tested concentration of 50 μM. The only polymers which had no12-amino-lauric acid residue in their sequence and which showedsignificant antimicrobial activity at lower concentrations wereC₈K(NC₈K)₅NH₂, C₈K(NC₈K)₆NH₂ and C₈K(NC₈K)₇NH₂ (SEQ ID NOs: 29-31,respectively) (see, entries 29-31 in column 6 of Table 3), whereaspolymers containing one or more of the more hydrophobic 12-amino-lauricacid residue, (see, entries 58-83 in Table 3), showed significantactivity at concentrations as low as 1.6 μM.

Evaluation of the effect of the hydrophobicity of the polymers in termsof the acetonitrile percentages of the HPLC mobile phase in which thepolymers were eluted further demonstrates the correlation between thisproperty and the antimicrobial activity of the polymer. As can be seenin the data presented in column 4 of Table 3, all the polymers whichdisplayed a significant level of antimicrobial activity against any oneof the tested bacteria were eluted in acetonitrile concentrations higherthan 36%, whereby none of the polymers that were eluted in acetonitrileconcentrations lower than 36% exhibited such an activity.

FIG. 1 presents the distribution of polymers which exhibited asignificant microbial activity (MIC value of less than 50 μM) in any oneof the four assays conducted. As is clearly seen in FIG. 1,antimicrobial activity against one or more of the tested bacteria wasexhibited only by polymers which were eluted at acetonitrileconcentrations of 36% and up and, furthermore, polymers which were foundactive against all the tested bacteria were eluted at acetonitrileconcentrations of 51% and up.

As can further be seen in Table 3, increasing the positive charge of thepolymers by increasing the number of the lysine residues in the polymerwas found to affect the antimicrobial activity of the polymers onlymarginally. Thus, polymers having net charges raging from +1 to +9 ineach series were tested for antimicrobial activity. The results,presented in Table 3, indicated, for example, that most of the polymerswith the highest net positive charge of +9, namely K(NC₄K)₇NH₂,NC₁₂K(NC₄K)₇NH₂, K(NC₈K)₇NH₂, NC₁₂K(NC₈K)₇NH₂, K(NC₁₂K)₇NH₂,(NC₁₂K)₈NH₂, (SEQ ID NOs: 14, 24, 38, 52, 64 and 71, respectively) (see,respective entries 14, 24, 38, 52, 64 and 71 in Table 3), did notexhibit significant activity, with only NC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 52)(see, entry 52 in Table 3) exhibiting significant activity.

As can be concluded from the data presented in Table 3, while none ofthe polymers series based on hydrophobic moieties containing 4-carbonchains inhibited bacterial proliferation, three polymers from thepolymers series based on hydrophobic moieties containing 8-carbon chainsinhibited growth of E. coli, displaying MIC values in the low micromolar(μg/ml) range. Several observations stem from the results shown in Table3: the equivalent 4-carbon chain based polymers, whose hydrophobicityvalues, represented by the percent of acetonitrile in the HPLC-RPgradient mobile phase at which the polymer was eluted and whichcorresponds to the estimated hydrophobicity of the polymer, variedbetween 20% and 25% (indicating low hydrophobicity) had virtually noantibacterial activity up to the highest concentration assayed (MIC>50μM). The more hydrophobic 8-carbon chain based polymers became activeonly when they reached hydrophobicity values of >40% (see, C₈K(NC₈K)₅NH₂(SEQ ID NO: 29), entry 29 in Table 3). Elongating the polymer by one C₈Ksubunit (see, C₈K(NC₈K)₆NH₂ (SEQ ID NO: 30), entry 30 in Table 3)increased the charge without significant change to hydrophobicity anddid not alter activity. Further addition of another C₈K subunit (see,C₈K(NC₈K)₇NH₂ (SEQ ID NO: 31), entry 31 in Table 3) had similar chargeand hydrophobicity effects but led to a two-fold enhanced activity.

Overall, these results indicate that an optimal antibacterial activityemerges when a polymer, as described herein, attains an optimal windowof charge and hydrophobicity, much as observed with conventional AMPs.

Table 4 below presents a summary of the results obtained in theseexperiments, in terms of the effect of the net positive charge of thepolymers and the antimicrobial activity thereof. Row 2 of Table 4presents the number of polymers in 9 bins, wherein each bin represents anet positive charge, starting from +9 to +1. Row 3 of Table 4 presentsthe total number of activity assays which were measured in the chargebin, namely, the number of polymers in the bin multiplied by the fourbacterial assays described above. Row 4 of Table 4 presents the numberof polymers in each of the bins that were found active against any oneof the four bacteria. Row 5 of Table 4 presents the percentage of theactive polymers from the total number of assays measured in the chargebin. As can be seen in Table 4, (row 4, for example), only a little ifany correlation between the net positive charge of the polymers andtheir antimicrobial activity was found. It appears from these resultsthat the only feature that seems to affect the antimicrobial activity ofthe tested polymers is a net positive charge that is greater than +1.

TABLE 4 Charge +9 +8 +7 +6 +5 +4 +3 +2 +1 Number of polymers 6 10 10 1214 16 15 11 1 Number of assays 24 40 40 48 56 64 60 44 4 Number ofactives 1 6 4 12 13 9 4 12 0 Percent actives 4.2% 15.0% 10.0% 25.0%23.2% 14.1% 6.7% 27.3% 0.0%

FIG. 2 presents the distribution of polymers according to the presentinvention which exhibited a significant microbial activity (MIC value ofless than 50 μM) in any one of the four assays mentioned above. As canbe clearly seen in FIG. 2, polymers which showed antimicrobial activityagainst any one of the four bacteria are scattered across the entirerange of charge values, excluding the +1 charge, and thus demonstratingthe lack of correlation between the net positive charge of the polymersand the antimicrobial activity thereof.

Overall, these results indicated that antibacterial activity emergedwhen a polymer attained an optimal window of charge and hydrophobicity,much as observed with conventional AMPs. These results also suggestedthat a parallel increase in hydrophobicity value might enhance potency.

Antimicrobial Activity Against Gram-Negative Bacteria

The activity of the novel polymers describes herein on Gram-negativebacteria has also been tested. To date, new antimicrobial agents thatare effective against Gram-negative bacteria are rarely found.

Table 5 below presents the results obtained in this study, and clearlyshows that C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplary polymer aspresented herein, displayed potent growth inhibitory activity against 9strains out of a panel of 10 Gram-negative bacterial strains.

“MIC” (column 3 in Table 5) represents the minimal inhibitoryconcentration of the exemplary polymer C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) inμM for each of the tested bacterial strains, which induced 100%inhibition of proliferation after 24 hours incubation. Values representthe mean from two independent experiments performed in duplicates.

As can be seen in Table 5, the MIC observed for C₁₂K(NC₈K)₇NH₂ (SEQ IDNO: 45) ranged from 1.6 μM to 12.5 μM and in general the MIC value formost bacterial strains was equal or inferior to 6.2 μM (14 μg/ml). Theseencouraging results were also observed for clinically challengingspecies such as Acinetobacter and Pseudomonas, for both of which a MICvalue of 3.1 was observed (see, entries 5 and 6 in Table 5respectively).

TABLE 5 Entry Gram-negative Bacteria Strain MIC 1 Enterobacter cloacaeCI 730 1.6 2 Brevundimonas diminuta ATCC 19146 1.6 3 Yersiniakristensenii ATCC 33639 1.6  4a Escherichia coli CI 3504 1.6*  4bEscherichia coli ATCC 25922 3.1* 5 Acinetobacter baumanii CI 1280 3.1 6Klebsiella pneumoniae CI 1286 3.1 7 Proteus mirabilis CI 1285 6.2 8Pseudomonas aeruginosa CI 8732 6.2 9 Stenotrophomonas maltophilia CI 74612.5 10  Serratia odorifera ATCC 33077 >50 *MIC determined using theClinical and Laboratory Standards Institute (CLSI) recommended procedureas presented hereinabove.

Development of Antimicrobial-Resistance in Bacteria:

The possible development of resistance to the polymers of the presentinvention was tested by measuring the MIC levels following multipleexposures of the bacteria to exemplary polymers according to the presentinvention, as described hereinabove in the Experimental Methods section.The tested polymers in these experiments were K(NC₁₂K)₃NH₂,C₁₂K(NC₈K)₅NH₂, C₁₂KKNC₁₂KNH₂ and C₁₂K(NC₈K)₇NH₂ (SEQ ID NOs: 60, 43, 78and 45 respectively), whereby the development of resistance of E. colito K(NC₁₂K)₃NH₂ and C₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 60 and 43 respectively)was compared with that of three classical antibiotics: gentamycin,tetracycline and ciprofloxacin, the development of resistance ofmethicilin-resistant S. aureus to C₁₂KKNC₁₂KNH₂ (SEQ ID NO: 78) wascompared with that of two classical antibiotics: rifampicin andtetracycline, and the development of resistance of E. coli toC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), evaluated during 15 serial passages, wascompared with that of three classical antibiotics, ciprofloxacin,imipenem, and tetracycline.

The data obtained in these experiments is presented in FIGS. 3a, 3b and3c . FIG. 3a presents the data obtained for K(NC₁₂K)₃NH₂ andC₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 60 and 45 respectively), FIG. 3b presentsthe data obtained for C₁₂KKNC₁₂KNH₂ (SEQ ID NO: 78) and FIG. 3c presentsthe data obtained for C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45).

As is clearly seen in FIG. 3a , the relative MIC value of K(NC₁₂K)₃NH₂and C₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 60 and 43 respectively) against E. coliremained stable for 10 successive subculture generations following theinitial exposure. In sharp contrast, during the same period of time, theMIC values tested with the reference antibiotic agents substantiallyincreased, reflecting the emergence of antibiotic-resistant bacteria.Thus, at the tenth generation, the MIC values increased by 4-fold fortetracycline and gentamycin, and by more than 16-fold for ciprofloxacin.These results demonstrate that exposing bacteria to the antimicrobialpolymers of the present invention do not result in development ofresistance.

As is clearly seen in FIG. 3b , the relative MIC value of C₁₂KKNC₁₂KNH₂(SEQ ID NO: 78) against methicilin-resistant S. aureus remained stablefor 15 successive subculture generations following the initial exposure.In sharp contrast, during the same period of time, the MIC values testedwith the reference antibiotic agents substantially increased, reflectingthe emergence of antibiotic-resistant bacteria. Thus, at the fifteenthgeneration, the MIC values increased by more than 230-fold forrifampicin, and by 4-fold for tetracycline. These results demonstrateagain that exposing bacteria to the antimicrobial polymers of thepresent invention do not result in development of resistance.

Similarly, it can be seen in FIG. 3c , the relative MIC value ofC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) against E. coli remained stable for 15successive subculture generations following the initial exposure. Insharp contrast, during the same period of time, the MIC values testedwith the reference antibiotic agents substantially increased, reflectingthe emergence of antibiotic-resistant bacteria. Thus, at the fifteenthgeneration, the MIC values increased by more than 60-fold forciprofloxacin, and by 8-fold for imipenem and tetracycline. Theseresults demonstrate yet again that exposing bacteria to theantimicrobial polymers of the present invention do not result indevelopment of resistance.

The development of antimicrobial resistance following exposure to thepolymers of the present invention was further evaluated in a crossresistance experiment, in which a methicilin-resistant strain of S.aureus was exposed to exemplary polymers of the present invention. Theresults obtained in this experiment are presented in Table 3hereinabove, under column “MIC MR S.a.” and clearly demonstrate thepersisting antimicrobial activity of the polymers of the presentinvention against an antibiotic-resistant bacteria, especially in thecase of C₁₂(KNC₁₂K)₂NH₂, C₁₂KKNC₁₂KNH₂, FmocK(NC₁₂K)₂, C₁₂K(KNC₁₂K)₂NH₂,C₁₂OrnNC₁₂OrnNH₂, C₁₂ArgNC₁₂ArgNH₂, C₁₂KNC₁₂KNH₂, C₁₂K(NC₁₂K)₂,C₁₂K(NC₈K)₄NH₂ and C₁₂KK(NC₈K)₄NH₂ (SEQ ID NOs: 79, 78, 95, 80, 84, 85,54, 87, 42 and 53, respectively) (see, respective entries 79, 78, 95,80, 84, 85, 54, 87, 42 and 53, in Table 3).

Kinetic Studies of Antimicrobial Activity at Time Intervals:

The kinetic rates of bactericidal activity of a representative polymerof the present invention, C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43), was tested asdescribed in the methods section above at concentrations correspondingto 3 and 6 times the MIC value. The results, presented in FIG. 4,clearly reflect the antibacterial activity of the polymer. As is shownin FIG. 4, the viable bacterial population was reduced by nearly sevenlog units within 6 hours upon being exposed to the polymer at aconcentration of 3 multiples of the MIC, and within 2 hours upon beingexposed to the polymer at a concentration of 6 multiples of the MIC.

FIG. 5 presents comparative plots demonstrating the kinetic bactericidaleffect of C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplary polymer accordingto the present invention, on E. coli. as compared with kineticbactericidal effect of various classical antibiotics as determined at aconcentration corresponding to six multiples of their respective MICvalue.

As can be seen in FIG. 5, C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), (blacktriangles) was responsible for rapid bacterial death namely,C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) reduced bacterial population from 106 to<50 CFU/ml within one hour compared to normal bacterial growth control(black circles), while Imipenem (white squares) and Ciprofloxacin (blacksquares) induced a weaker bactericidal effect, and Tetracycline (whitecircles) was merely bacteriostatic. The plotted values represent themean±standard deviations obtained from at least two independentexperiments. The stared datum points indicate that bacteria were notdetected at the minimum level of sensitivity (<50 CFU/ml).

These remarkable results further demonstrate the efficacy of theantimicrobial polymers of the present invention, in terms of anefficient pharmacokinetic profile.

Antimicrobial Activity at Enhanced Outer-Membrane PermeabilityConditions:

The results obtained following addition of the cation-chelator EDTA tothe assay buffer, which was aimed at enhancing the outer-membranepermeability of gram-negative bacteria such as E. coli, are presented inTable 3 above under the column headed “MIC EDTA E.c.”. These resultsclearly show that the activity profile of the polymers in the presenceof EDTA is different than that obtained without EDTA (presented in Table3 above, column headed “MIC E.c.”). Thus, polymers such as K(NC₈K)₇NH₂,NC₁₂K(NC₈K)₄NH₂, NC₁₂K(NC₈K)₅NH₂, C₁₂K(NC₁₂K)₂NH₂, K(NC₁₂K)₅NH₂,K(NC₁₂K)₆NH₂, K(NC₁₂K)₇NH₂, (NC₁₂K)₄NH₂, (NC₁₂K)₅NH₂, (NC₁₂K)₆NH₂,(NC₁₂K)₇NH₂ and (NC₁₂K)₈NH₂ (SEQ ID NOs: 38, 49, 50, 55, 62, 63, 64, 67,68, 69, 70 and 71, respectively), which exhibited minor or noantimicrobial activity in the absence of EDTA, became up to more than 50folds more active in its presence (see, respective entries 38, 49, 50,55, 62, 63, 64, 67, 68, 69, 70 and 71, in Table 3). Other polymers, suchas K(NC₁₂K)₄NH₂, C₈K(NC₈K)₅NH₂, C₁₂K(NC₈K)₄NH₂, NC₁₂K(NC₈K)₆NH₂ and(NC₁₂K)₃NH₂ (SEQ ID NOs: 61, 29, 42, 51 and 66, respectively), whichexhibited only marginal antimicrobial activity in the absence of EDTA,became between 11-folds and 4-fold more active in its presence,respectively (see, respective entries 61, 29, 42, 51 and 66 in Table 3).

These results illuminate the tight correlation between membranepermeability of antimicrobial agents and their efficacy and furtherdemonstrate the complex relationship and delicate balance between thepositive charge and the hydrophobic characteristics of the polymers ofthe present invention on the antimicrobial activity thereof.

Susceptibility to Plasma Proteases Assays Results:

The susceptibility of the polymers of the present invention to enzymaticcleavage was assessed by pre-incubating exemplary polymers according tothe present invention, C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43), K(NC₁₂K)₃NH₂ (SEQID NO: 60), C₁₂KNC₁₂KNH₂ (SEQ ID NO: 54), and C₁₂KKNC₁₂KNH₂ (SEQ ID NO:78), and an exemplary reference AMP, a 16-residues dermaseptin S4derivative (S4₁₆) (SEQ ID NO: 102), in human plasma (50%) for varioustime periods and thereafter determining the antibacterial activitythereof against E. coli and S. aureus. Statistical data were obtainedfrom at least two independent experiments performed in duplicates.

The results are presented in Table 6 hereinbelow, wherein “MIC (E.c.)C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43) (μM)” is the minimal inhibitoryconcentration in μM of C₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43), as measured forE. coli; “MIC (E.c.) K(NC₁₂K)₃NH₂ (SEQ ID NO: 60) (μM)” is the minimalinhibitory concentration in μM of K(NC₁₂K)₃NH₂ (SEQ ID NO: 60), asmeasured for E. coli; “MIC (E.c.) S4₁₆ (SEQ ID NO: 102) (μM)” is theminimal inhibitory concentration in μM of S4₁₆, an exemplary dermaseptinserving as a reference AMP, as measured for E. coli; “MIC S.a.C₁₂KNC₁₂KNH₂ (SEQ ID NO: 54) (μM)” is the minimal inhibitoryconcentration in μM of C₁₂KNC₁₂KNH₂ (SEQ ID NO: 54), as measured for S.aureus; and “MIC (S.a.) C₁₂KKNC₁₂KNH₂ (SEQ ID NO: 78) (μM)” is theminimal inhibitory concentration in μM of C₁₂KKNC₁₂KNH₂ (SEQ ID NO: 78),as measured for S. aureus.

TABLE 6 MIC (E.c.) MIC (E.c.) MIC (E.c.) MIC (S.a.) MIC (S.a.)Incubation C₁₂K(NC₈K)₅NH₂ K(NC₁₂K)₃NH₂ S4₁₆ C₁₂KNC₁₂KNH₂ C₁₂KKNC₁₂KNH₂time (SEQ ID NO: 43) (SEQ ID NO: 60) (SEQ ID NO: 98) (SEQ ID NO: 54)(SEQ ID NO: 78) (hours) (μM) (μM) (μM) (μM) (μM) 0 3.1 12.5 3.1 12.5 3.13 3.1 12.5 >50 12.5 3.1 6 3.1 12.5 >50 25 6.3 18 3.1 12.5 >50 25 6.3

As is shown in Table 6, while the reference AMP, S4₁₆, was completelyinactivated upon exposure to human plasma, the polymers of the presentinvention maintained their activity, and thus, the superior stability ofthe polymers according to the present invention as compared with that ofthe highly active yet unstable AMPs was clearly demonstrated. Morespecifically, as is shown in Table 6, the dermaseptin S4₁₆ did notdisplay a measurable MIC after 3 hours exposure to serum enzymes, evenat a concentration of more than 16-folds higher (greater than 50 μM)than the MIC value, indicating that the peptide was inactivated probablydue to enzymatic proteolysis.

In sharp contrast, the polymers of the present invention exhibitedprolonged resistance to enzymatic degradation. As is further shown inTable 6, the activity of short polymers such as C₁₂KNC₁₂KNH₂ andC₁₂KKNC₁₂KNH₂ (SEQ ID NOs: 54 and 78) was reduced only by 2-folds after6 hours exposure to plasma enzymes while longer polymers such asK(NC₁₂K)₃NH₂ and C₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 60 and 43) did not displayany degree of inactivation even after 18 hours incubation.

Hemolysis Assays:

The toxic hemolytic effect of the polymers of the present invention onhuman erythrocytes (red blood cells, RBC) was assayed as describedhereinabove. The results are presented in Table 3, under the columnheaded “LC₅₀”, in terms of the lytic concentrations that induced 50%(LC₅₀) lysis of red blood cells in phosphate buffer (PBS).

As shown in Table 3, polymers such as C₁₂K(NC₈K)₇NH₂, C₈(KNC₁₂K)₂NH₂,C₈K(KNC₁₂K)₂NH₂, NC₁₂K(KNC₁₂K)₂NH₂, C₁₂K(NC₈K)₅NH₂, C₁₂K(NC₈K)₆NH₂,NC₁₂(KNC₁₂K)₂NH₂, C₁₂KK(NC₈K)₄NH₂ and K(NC₁₂K)₃NH₂ (SEQ ID NOs: 45, 76,77, 83, 43, 44, 82, 53 and 60, respectively) (see, respective entries45, 76, 77, 83, 43, 44, 82, 53 and 60 in Table 3) which exhibited highantimicrobial activity, displayed low hemolytic activity. As is furthershown in Table 3, polymers including various fatty acid moietiesconjugated to the N-terminus thereof and/or a relatively large number oflysine residues, were particularly found to exhibit potent antibacterialactivity along with low hemolytic activity. These results clearlydemonstrate the low toxicity of the polymers of the present inventionagainst human red blood cells.

FIG. 6 presents comparative plots demonstrating the hemolytic effect ofC₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplary polymer as presentedherein, as compared to the hemolytic effect of bivalirudin, a synthetic20 amino acid peptide, which is clinically used as a specific andreversible direct thrombin inhibitor approved by FDA for intravenousadministration, and to the hemolytic effect of MSI-78, a magaininderivative that was recently assessed in human clinical trials fortreatment of diabetic foot ulcers, determined against human RBC (10%hematocrit) after 1 hour incubation at 37° C. in presence of threepolymer/peptide concentrations, namely 31 μM (striped bars), 94 μM (graybars) and 156 μM (white bars). Plotted values represent themean±standard deviations obtained from at least four independentexperiments.

As can be seen in FIG. 6, C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) did notexhibited any hemolytic activity when tested against human red bloodcells at all three experimental concentrations, and did not yield thecharacteristic dose-response profile observed with conventional AMPs.Both bivalirudin, a non-antimicrobial peptide, and C₁₂K(NC₈K)₇NH₂ (SEQID NO: 45) displayed merely a “background level” activity at least up to156 μM, a concentration that corresponds to about 100 folds the MICvalue of C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) against various bacteria.Contrarily, MSI-78 (SEQ ID NO: 96) exhibited a high degree of hemolysisat concentrations as low as 31 μM and 94 μM.

Circular Dichroism (CD):

The secondary structure of selected polymers according to the presentinventions was studied by circular dichroism (CD) measurements invarious media, as described hereinabove in the Experimental Methodssection. The CD profiles of C₁₂K(NC₈K)₅NH₂ and C₁₂K(NC₈K)₇NH₂ (SEQ IDNO: 43 and 45), exemplary antimicrobial polymers according to thepresent invention, and NC₁₂K₄S4₍₁₋₁₄₎ (SEQ ID NO: 101), an exemplarydermaseptin derivative, are presented in FIGS. 7 and 8. The CD datapresented represent an average of three separate recordings values.

FIG. 8 presents the circular dichroism spectra of C₁₂K(NC₈K)₇NH₂ (SEQ IDNO: 45), an exemplary polymer as presented herein (gray lines), andcontrol antimicrobial peptide K₄S₄(1-16) (SEQ ID NO: 98) (black lines),taken in PBS alone (dashed lines) or in presence of 2 mM POPC:POPG (3:1)liposomes suspended in PBS (solid lines) (data represent average valuesfrom three separate recordings).

As is shown in FIG. 7 and FIG. 8, the CD spectra of the polymers of thepresent invention displayed a minimum near 200 nm, indicating a randomstructure. The same CD spectra were observed in assays conducted in thepresence and absence of liposomes. The CD spectra of the controldermaseptin NC₁₂K₄S4₍₁₋₁₄₎ (SEQ ID NO: 101) and control antimicrobialpeptide K₄S₄(1-16) (SEQ ID NO: 98) showed a typical spectrumcharacteristic of an alpha-helical secondary structure. Similar resultswere observed in 20% trifluoroethanol/water (data not shown). Ingeneral, secondary structure imparts a distinct CD to their respectivemolecules. Therefore, the alpha helix and beta sheet typically observedin polypeptides and proteins have CD spectral signatures representativeof their structures. The lack of these characteristic CD spectralsignatures representative of a secondary structure elements in thespectra obtain for the polymers presented herein is indicative of their“random” secondary structure, or lack thereof.

Surface Plasmon Resonance Assay:

The binding properties of exemplary polymers according to the presentinvention to membranes were studied using surface plasmon resonance(SPR) measurements, as described hereinabove in the Methods section.

The obtained data indicated that the polymers according to the presentinvention display high affinity binding to a model membrane mimickingthe bacterial plasma membrane, with K_(app) ranging from 10⁴ to 10⁷M⁻¹). FIG. 9, for example, presents the data obtained withC₁₂K(NC₈K)₅NH₂ (SEQ ID NO: 43), and demonstrates the high affinitybinding of this exemplary polymer according to the present invention(K_(app) of 9.96×10⁴ M⁻¹ to a model membrane.

An additional exemplary antimicrobial polymer according to the presentinvention, K(NC₁₂K)₃NH₂ (SEQ ID NO: 60), displayed an even higheraffinity binding (K_(app) of 6.3×10⁵ M⁻¹, data not shown).

These results substantiate the affinity of the polymers of the presentinvention towards the membranes of a pathogenic microorganism.

Lipopolysaccharide Binding Assay:

The binding affinity of the positively charged polymers according to thepresent invention to the negatively charged lipopolysaccharides (LPS)present on the membrane of gram-negative bacteria was measured asdescribed in the Methods section hereinabove. The maximal binding levelsof seven exemplary polymers according to the present invention,KNC₈KNH₂, K(NC₈K)₂NH₂, K(NC₈K)₃NH₂, K(NC₈K)₆NH₂, KNC₁₂KNH₂, K(NC₁₂K)₂NH₂and K(NC₁₂K)₃NH₂ (SEQ ID NOs: 32, 33, 34, 37, 58, 59 and 60respectively), to liposomal membranes before and after incubation withLPS, as measured in these assays, are presented in FIG. 10.

As can be seen in FIG. 10, the binding affinity of a polymer to themembrane is affected by the length of the polymer. Thus, for example,the binding affinity of K(NC₈K)₆NH₂ (SEQ ID NO: 37) is higher than thatof KNC₈KNH₂ (SEQ ID NO: 32) and the binding affinity of K(NC₁₂K)₃NH₂(SEQ ID NO: 60) was found higher than that of KNC₁₂KNH₂ (SEQ ID NO: 58).

As can be further seen in FIG. 10, the same correlation between thepolymer length and its binding affinity to LPS was observed. Thus, forexample, the polymers K(NC₈K)₆NH₂ (SEQ ID NO: 37) and K(NC₁₂K)₃NH₂ (SEQID NO: 60) each exhibits close to 2-fold reduction of affinity toliposomal membrane following incubation with LPS, indicating binding ofthe polymers to LPS during the incubation period, which interferes withtheir binding to the membranal liposomes.

These results provide further support to a mechanism of action of thepolymers that involves strong interaction with LPS, which promotes adestructive action against the bacterial membrane and by which the riskof development of endotoxemia is reduced.

DNA Binding Assay:

The binding properties of exemplary polymers according to the presentinvention to nucleic acids were studied by determining their ability toretard migration of DNA plasmids during gel electrophoresis in a 1%agarose gel.

The obtained results show that the polymers according to the presentinvention retard the migration of various plasmids (e.g., pUC19, pGL3Luciferase Reporter Vector (Promega)) in a dose dependent manner.Representative results, obtained with the plasmid pUC19 in the absenceand presence of three exemplary polymers of the present invention,C₁₂KKNC₁₂KNH₂, K(NC₄K)₇NH₂ and C₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 78, 14 and 60respectively), are presented in FIG. 11 (Note: isolation of the plasmidfrom a bacterial culture results in three major bands and several minorbands, as seen in the leftmost slot of the gel's UV image). An apparentdose-dependent behavior was evident in the presence of the shortesttested polymer C₁₂KKNC₁₂KNH₂ (SEQ ID NO: 78). The dose-dependentbehavior was further accentuated with the longer tested polymersK(NC₄K)₇NH₂ and C₁₂K(NC₈K)₅NH₂ (SEQ ID NOs: 14 and 60 respectively).Thus, at the lowest dose of C₁₂KKNC₁₂KNH₂ (SEQ ID NO: 78) (polymer toDNA ratio of 1:1), the supercoiled plasmid DNA band disappeared whereasthe other bands displayed a smeared pattern. These results suggest thatthe inhibitory effect of the polymers of the present invention is higherwith supercoiled DNA. Increasing the polymer doses resulted inaccentuated effect, such that the retardation effect extended to all DNAspecies.

Furthermore, it was found that various polymer-DNA complexes remainedintact after exposure to either DNAse digestive enzymes or peptidasedigestive enzymes. These findings reveal a tight binding between thepolymers of the present invention and the DNA molecule, exhibited by themutual shielding exerted by the polymers to the DNA molecules and viceversa.

Saliva Microbicidal Assays:

The antimicrobial activity of an exemplary polymer of the presentinvention, C₈K₈ (SEQ ID NO: 100), against microorganisms in human salivawas studied as described above. FIG. 12 presents the results obtained inthis study in terms of the logarithmic units of CFU per ml as a functionof the incubation time of the samples with the vehicle buffer (control),IB-367 (SEQ ID NO: 97) (antimicrobial agent with known activity control)and C₈K₈ (SEQ ID NO: 100). The results show that while in the control,untreated group the saliva microorganisms are persistent and proliferatewithout any treatment, the growth of saliva microorganisms treated isinhibited but proliferation is resumed after 30 minutes; whereby thegrowth of the saliva microorganisms treated with the polymer accordingto the present invention is inhibited without recovery.

Anti-Malarial Assays:

A of a group of polymers, according to the present invention, weretested for their anti-malarial effect on parasite growth and onmammalian cells. The obtained results are presented in Table 7 below,wherein:

“IC50 parasite (μM)” represents the concentration of the tested polymerin μM that is required for 50% inhibition of the growth of the malariacausing parasites, measured as described hereinabove (column 3 in Table7);

“IC50 MDCK (μM)” represents the concentration of the tested polymer inμM that is required for 50% inhibition of growth of MDCK cells, measuredas described hereinabove (column 3 in Table 7); and

“IC50 Ratio” represents the ratio of IC50 MDCK over IC50 parasite,indicating the specificity of the polymer to parasitic membranes overthat of mammalian cells.

TABLE 7 IC₅₀ IC₅₀ Entry parasite MDCK IC₅₀ (SEQ ID NO:) Polymer (μM)(μM) Ratio A (54) C₁₂KNC₁₂KNH₂ 3.54 156.8 44.29 B (65) (NC₁₂K)₂NH₂ 4.63609.2 131.58 C (55) C₁₂K(NC₁₂K)₂NH₂ 0.85 92.1 108.35 D (66) (NC₁₂K)₃NH₂0.14 48.3 352.55  E (56) C₁₂K(NC₁₂K)₃NH₂ 0.08 37.3 449.40  F (67)(NC₁₂K)₄NH₂ 1.59 57.0 35.85 G (58) KNC₁₂KNH₂ 68.20 693.8 10.17 H (59)K(NC₁₂K)₂NH₂ 7.85 157.4 20.05  I (60) K(NC₁₂K)₃NH₂ 1.72 347.0 201.74

As shown in Table 7, some of the polymers have shown very high activityagainst malarial parasites having an IC₅₀ in the sub-micromolar range,as presented in the column denoted IC₅₀ parasite (μM) (see entries D andE in Table 7 above). The structure-activity relationship conclusion thatemerges from this series is that lengthening of the chain increases theanti-malarial activity (reduces the IC₅₀). The presence of the alkylmoiety at the N-terminus of the lysine, invariably increases theanti-malarial activity (see, entries G and A, entries H and C andentries I and E in Table 7 above). For some polymers, the amino alkyladds further activity (see, entries C and D in Table 7 above) but thisperformance is not always consistent (see, entries A and B and entries Eand F in Table 7 above).

There are similar consistencies for the effect of the polymers of thepresent invention on the MDCK cells. Addition of an alkyl at theN-terminus of the lysine results in a decrease in activity (see, entriesG and A, entries H and C, and entries I and E in Table 7 above). Theamino alkyl moiety usually results in decreased activity (see, entries Aand B, and entries C and D in Table 7 above), but the opposite effectwas observed for the longest polymers (see, entries E and F in Table 7above).

The ratio of IC₅₀ is essentially equivalent to the therapeutic ratio.Thus, entries D and E in Table 7 above show the most therapeuticallyefficient polymers, according to the present invention.

Similar results were obtained with the primary cultures ofcardio-fibroblasts (CF) and HepG2 transformed cells (results not shown).

Another series of polymers was tested for anti-malarial activity inorder to further investigate the structure-activity relationship withrespect to polymer length and hydrophobic moiety residue length.

The results, presented in Table 8 below, wherein “IC50 (μM)” representsthe concentration of the tested polymer in μM that is required for 50%inhibition of the growth of the malaria causing parasites, measured asdescribed hereinabove (column 3 in Table 8), indicate that the additionof caprylic acid (C₈) to the N-terminus of the lysine residue increasesthe anti-malarial potency considerably (up to 67 fold), but thisamplification diminishes as the chain length increases. Substitution ofC₈ with lauric acid (C₁₂) results in a further increase theanti-malarial potency (up to 20-fold), whereas further substitution atthis terminus with ω-aminolauric acid (NC₁₂) reverts the potencyconsiderably.

Among the most active polymers in the C₁₂K(NC₈K)_(n)NH₂ (SEQ ID NOs:39-45) group, the anti-malarial potency diminishes with increase polymerlength (see, entries 15-21 in Table 8 below). The opposite trend wasobserved for the non-acylated (at the N-terminus) group K(NC₈K)_(n)NH₂(SEQ ID NOs: 32-38) (see, entries 1-7 in Table 8 below) although theyexhibit an overall lower activity. No such consistent trends could beobserved for the other groups.

None of the polymers of this series caused lysis of infected RBC atconcentrations that are at least 2-fold higher than their respectiveIC₅₀ (data not shown).

TABLE 8 Entry IC₅₀ (SEQ ID NOs:) Polymer (μM)  1 (32) KNC₈KNH₂ 260  2(33) K(NC₈K)₂NH₂ 180  3 (34) K(NC₈K)₃NH₂ 130  4 (35) K(NC₈K)₄NH₂ 90  5(36) K(NC₈K)₅NH₂ 84  6 (37) K(NC₈K)₆NH₂ 71  7 (38) K(NC₈K)₇NH₂ 47  8(25) C₈KNC₈KNH₂ 16  9 (26) C₈K(NC₈K)₂NH₂ 2.7 10 (27) C₈K(NC₈K)₃NH₂ 14.811 (28) C₈K(NC₈K)₄NH₂ 48.9 12 (29) C₈K(NC₈K)₅NH₂ 44.1 13 (30)C₈K(NC₈K)₆NH₂ 30.5 14 (31) C₈K(NC₈K)₇NH₂ 37.2 15 (39) C₁₂KNC₈KNH₂ 0.3816 (40) C₁₂K(NC₈K)₂NH₂ 0.2 17 (41) C₁₂K(NC₈K)₃NH₂ 1.16 18 (42)C₁₂K(NC₈K)₄NH₂ 2.43 19 (43) C₁₂K(NC₈K)₅NH₂ 5.57 20 (44) C₁₂K(NC₈K)₆NH₂9.9 21 (45) C₁₂K(NC₈K)₇NH₂ 15.8 22 (46) NC₁₂KNC₈KNH₂ 120.1 23 (47)NC₁₂K(NC₈K)₂NH₂ 99.5 24 (48) NC₁₂K(NC₈K)₃NH₂ 93.7 25 (49)NC₁₂K(NC₈K)₄NH₂ 72.9 26 (50) NC₁₂K(NC₈K)₅NH₂ 70.6 27 (51)NC₁₂K(NC₈K)₆NH₂ 66.5 28 (52) NC₁₂K(NC₈K)₇NH₂ 89.8

The anti-malarial effect of the polymer C₁₂K(NC₁₂K)₃NH₂ (SEQ ID NO. 56)(see, entry E in Table 7 above) has been tested by exposing parasitecultures at the ring and the trophozoite stages for various lengths oftime and different polymer concentrations, the polymer has then beenremoved and after 48 hours all cultures that were subjected for thedifferent treatments were tested for parasite viability using thehypoxanthine incorporation test.

The IC₅₀ for each treatment has been calculated for thechloroquine-resistant FCR3 strain versus chloroquine-sensitive NF54strain, and the results are presented in FIG. 13. As seen in FIG. 13 thering stage is more sensitive to the polymer than the trophozoite stagewhere it also takes a longer time to exert the inhibitory action. Italso seems that the effect is cumulative in that the IC₅₀ values at 48hours are lower than those observed with shorter exposure times.

The effect of time of exposure of parasite cultures to C₁₂KNC₈KNH₂ (SEQID NO. 39) (see, entry 15 in Table 8 above) at different stages onparasite viability is shown in FIG. 14. As can be seen in FIG. 14, theresults indicate that ring and trophozoite stages are almost equallysensitive to C₁₂KNC₈KNH₂ (SEQ ID NO: 39), yet a period of 24 hours isrequired in order to exert the full inhibitory activity on the rings andmore so for the trophozoites stage.

In-Vivo Therapeutic Efficacy:

The therapeutic efficacy of C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) was assessedusing a murine peritonitis-sepsis model after intraperitoneal infectionwith E. coli and intraperitoneal treatment with the tested and controlagents one hour post-infection.

FIGS. 15a-b present the rate of survival, monitored over a time periodof 7 days, of infected mice (n=10 per group) inoculatedintraperitoneally with 2.5×10⁶ CFUs of E. coli CI 3504 (FIG. 15a ) and5×10⁶ CFUs of E. coli CI 3504 (FIG. 15 b), and subsequently treated onehour after infection by intraperitoneal administration of PBS (blackcircles), a single dose of 4 mg/kg C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45) (graysquares) or four doses of 2 mg/kg imipenem at (asterisk).

As can be seen in FIGS. 15a-b , the polymer C₁₂K(NC₈K)₇NH₂ (SEQ ID NO:45) significantly prevented mortality of mice infected with twodifferent lethal inocula. In these representative experiments, at thelow dose inoculum (2.5×10⁶ CFU/mouse), survival of infected mice treatedwith polymer was 100% compared to 20% in the vehicle treated controlgroup (p<0.005). At the higher dose inoculum (5×10⁶ CFU/mouse), survivalwas 80%, compared to 0% survival in the vehicle-treated group (p<0.005).Treatment with a classical antibiotic (imipenem, 4 doses during 28hours, starting one hour after infection) resulted in survival of 100%and 90% in the two inocula studied, respectively. Overall, these resultsclearly demonstrate the beneficial therapeutic potential use of thepolymers presented herein, and their efficacy in the treatment of harshbacterial infections.

The results obtained in this study, which reflect a comprehensiveefficacy profile, demonstrate that C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), apolymer according to the present invention, although displaying lessefficacious MIC levels (MIC E. coli of 3.1) as compared to certainclassical antibiotic agents, such as imipenen (MIC E. coli of 0.6), thepolymer still proves a more efficacious antimicrobial agent.

In-Vivo Toxicity:

In-vivo acute toxicity of the polymers presented herein was examined byintraperitoneal injection of 0 μg (blank control), 100 μg, 250 μg, and500 μg of freshly prepared C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45), an exemplarypolymer according to the present invention, to groups of 12 mice; thusthe dosage corresponding to 0, 4, 10, and 20 mg/kg of body weight.Animals were directly inspected for adverse effects for 4 hour, andmortality was monitored for 7 days thereafter.

FIG. 16 presents the rate of survival, monitored over time period of 6days, of female ICR mice (n=12 per group) treated intraperitoneally with0 mg/kg of body weight (white bars) 4 mg/kg of body weight (sparselystriped bars), 10 mg/kg of body weight (densely striped bars) and 20mg/kg of body weight (black bars) of C₁₂K(NC₈K)₇NH₂ (SEQ ID NO: 45).

As can be seen in FIG. 16, only 25% of the mice treated with the highestdose of 20 mg/kg of body weight died, while all the mice treated with 4and 10 mg/kg of body weight survived throughout the duration of theexperiment.

Based on the experimental results presented herein, the polymersaccording to the present invention offer several advantages overconventional AMPs, which are mostly of limited utility as therapeuticagents due to their low bioavailability and/or high toxicity. Frompharmacologic, therapeutic and other practical points of view, thepolymers presented herein represent a novel and promising family ofantimicrobial agents that are devoid of AMPs intrinsic disadvantages.They may therefore be beneficially utilized in various antimicrobialfields including the treatment of medical conditions associated withpathogenic microorganisms.

Moreover, the inherently simple and incremental nature in designingpolymer libraries provides a new alternative and a systematic tool forthe dissection of the relative importance of charge and hydrophobicity,the parameters believed to be most crucial to antimicrobial activity andtheir role in selective activity.

The peptide-like backbone, the physico-chemical characteristics, thebroad antibacterial activity spectrum, the rapid bactericidal kineticsand the bacterial challenge in developing resistance, demonstratedherein for the polymers of the present invention, are comparable andeven superior to those of conventional AMPs and thus reminiscent oftheir postulated membrane disruptive properties and other eventualtargets. In this respect, the results presented herein may suggest thata defined secondary structure does not necessarily play a determiningrole. Rather, activity appears to depend substantially on a subtleinterplay between positive charge and hydrophobicity.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

What is claimed is:
 1. A polymer comprising a plurality of positivelycharged amino acid residues and at least one hydrophobic moiety residue,wherein at least one of said at least one hydrophobic moiety residue isbeing covalently linked to at least two amino acid residues in saidplurality of amino acid residues via the N-alpha of one amino acidresidue and via the C-alpha of the other amino acid residue in said atleast two amino acid residues.
 2. The polymer of claim 1, wherein saidplurality of amino acid residues comprises from 2 to 50 amino acidresidues.
 3. The polymer of claim 1, wherein said positively chargedamino acid residues are selected from the group consisting of lysineresidues, histidine residues, ornithine residues, arginine residues andcombinations thereof.
 4. The polymer of claim 1, wherein said at leastone hydrophobic moiety has a carboxylic group at one end thereof and anamine group at the other end thereof.
 5. The polymer of claim 4, whereinsaid at least one hydrophobic moiety residue is linked to each of saidat least two amino acid residues via a peptide bond.
 6. The polymer ofclaim 1, comprising from 1 to 50 hydrophobic moiety residues.
 7. Thepolymer of claim 1, wherein said at least one hydrophobic moiety residuecomprises a fatty acid residue.
 8. The polymer of claim 1, wherein eachof said at least one hydrophobic moiety is an ω-amino-fatty acidresidue.
 9. The polymer of claim 1, comprising at least two hydrophobicmoiety residues, wherein at least one of said at least two hydrophobicmoiety residues is being linked to the N-alpha of an amino acid residueat the N-terminus of said plurality of amino acid residues.
 10. Thepolymer of claim 3, comprising 2 to 50 lysine residues and 1 to 50ω-amino-fatty acid residues, wherein: at least one of said ω-amino-fattyacid residues is covalently linked to at least two of said lysineresidues via the N-alpha of one lysine residue and via the C-alpha ofthe other lysine residue in said at least two lysine residues, said atleast one ω-amino-fatty acid is linked to each of said lysine residuesvia a peptide bond, and wherein: each of said lysine residues isindependently selected from the group consisting of a lysine residue anda lysine residue having a fatty acid residue linked to a side-chainthereof; an N-terminus of the polymer is selected from the groupconsisting of a lysine residue, an ω-amino-fatty acid residue linked viaa peptide bond to a lysine residue and a fatty acid residue linked via apeptide bond to said lysine residue; and a C-terminus of the polymer isselected from the group consisting of a lysine residue and a lysineresidue terminated with an amide group.
 11. The polymer of claim 10,wherein said ω-amino-fatty acid residue is selected from the groupconsisting of 4-amino-butyric acid residue, 6-amino-caproic acidresidue, 8-amino-caprylic acid residue, 10-amino-capric acid residue,12-amino-lauric acid residue, 14-amino-myristic acid residue,16-amino-palmitic acid residue, 16-amino-palmitoleic acid residue,18-amino-stearic acid residue, 18-amino-oleic acid residue,18-amino-linoleic acid residue, 18-amino-linolenic acid residue and20-amino-arachidonic acid residue.
 12. The polymer of claim 11, havingan amino acid sequence selected from the group consisting of the aminoacid sequences as set forth in SEQ ID NOs: 1-83 and 86-94 as set forthherein.
 13. The polymer of claim 12, each of said ω-amino-fatty acidresidues covalently linked to said at least two of said lysine residuesis 8-amino-caprylic acid residue.
 14. The polymer of claim 1, beingselected from the group of compounds presented in Table
 3. 15. Apharmaceutical composition comprising, as an active ingredient, thepolymer of claim 1 and a pharmaceutically acceptable carrier.
 16. Thepharmaceutical composition of claim 15, further comprising at least oneadditional therapeutically active agent.
 17. The pharmaceuticalcomposition of claim 16, wherein said at least one additionaltherapeutically active agent comprises an antibiotic agent.
 18. Animaging probe for detecting a pathogenic microorganism, the imagingprobe comprising the polymer of claim 1, said polymer further comprisingat least one labeling agent attached thereto.